BR102018069308A2 - RECOMBINANT YELLOW CELL, METHODS FOR PRODUCING RECOMBINANT YELLOW CELL AND HYDROXYLE COLLAGEN, COLLAGEN, AND BIOFABRICATED LEATHER MATERIAL. - Google Patents

Abstract

This invention relates to genetically engineered yeast strains and methods for producing recombinant protein (e.g. collagen). The recombinant protein of the present invention is used to produce biofabricated leather or a material with leather-like properties containing recombinant or engineered collagen. yeast strains are engineered to produce ascorbate and / or increased α-ketoglutarate production.

Description

[001] This application relates to U.S. patent application no.

15 / 433,566 titled Biofabricated Material Containing Collagen Fibrils, 15 / 433,650, titled Method for Making a Biofabricated Material Containing Collagen Fibrils, 62 / 526,912 titled Recombinant Yeast Strains and 62 / 539,213 titled Yeast Strains and Method for Controlling Hydroxylation of Recombinant Collagen, and priority of claims for 62 / 562,109, filed on September 22, 2017, entitled Recombinant Yeast Strains. All content of these requests is incorporated into this document by reference.

Field Of Invention [002] The invention in this document provides genetically engineered yeast strains and methods for producing recombinant proteins and carbohydrates. In one embodiment, the invention in this document provides genetically engineered strains of yeast and methods for producing recombinant collagen that can be used to produce biofabricated leather materials, articles comprising biofabricated materials and / or material with similar properties to manipulated or recombinant collagen leather. . Yeast strains are manipulated to produce increased amounts of hydroxylated protein (eg, collagen) and carbohydrates by improving the hydroxylation reaction through the use of an α-ketoglutarate (kgtP) transporter and / or one or more polypeptides ascorbate synthesis pathway, such as GDP-L-Gal phosphorylase, phosphate inositol phosphate, GDP-Mannose-3,5-epimerase and / or L-gulono-1,4lactone oxidase, aldonolactonase, glucuron lactone reductase, D- glucuronate

Petition 870180132932, of 9/21/2018, p. 10/93 / 80 reductase, uronolactonase, D-glucuronine kinase, glucuronate-1-phosphate uridylyltransferase, UDP-D-glucose dehydrogenase, UTP-glucose-1-phosphate uridylyltransferase, phosphoglucomutase and / or hexokinase.

Description of Related Art [003] Leather is used in a wide variety of applications, including upholstery of furniture, clothing, shoes, bags, bags, accessories and automotive applications. The estimated global commercial value in leather is approximately US $ 100 billion per year (Future Trends in the World Industry and Trade in Leather Products, United Nations Industrial Development Organization, Vienna, 2010) and there is a continuous and growing demand for leather products. Traditional leather production wastes a lot when it comes to animals and chemicals. The increasing demand for animal skins to keep up with the continuous and growing demand for leather products puts additional pressure on our increasingly outdated food sources, while chemical waste continues to contribute to the erosion of our environmental conditions. Thus, new ways are needed to meet this demand in view of the economic, environmental and social costs of leather production. To keep up with technological and aesthetic trends, producers and users of leather products are looking for new materials that exhibit superior strength, uniformity, processability and elegant and attractive aesthetic properties that incorporate natural components.

[004] Given the population growth and the global environment, there will be a need for alternative materials that have a similar aesthetic to leather and improved functionality. Leather is animal skin and consists almost entirely of collagen. There is a need for new sources of collagen that can be incorporated into biofabricated materials.

[005] The production of biofabricated materials using recombinantly expressed collagen faces a series of challenges, including the

Petition 870180132932, of 9/21/2018, p. 11/93 / 80 need for a method to efficiently produce collagen in the forms and quantities necessary for various commercial applications. For some applications, a softer and more permeable collagen component is desired; in others, a harder, stronger and more durable collagen component is needed.

[006] The inventors sought to address these challenges by manipulating recombinant yeasts with a kgtP gene that provides an α-ketoglutarate transporter and / or with one or more genes that allow an ascorbate synthesis pathway to function so that the recombinant yeast produces ascorbate.

Summary of the Invention [007] One aspect of the invention is directed to modified yeast cells that contain an α-ketoglutarate transporter. The modification includes the transformation of a kgtP gene in yeast that allows the yeast to produce the α-ketoglutarate transporter. Yeast cells can be further modified to express and / or overexpress a protein that hydroxyl proline and / or lysine residues to yield hydroxyproline, 3-hydroxyproline (Hyp) and hydroxylisin (Hyl) and glycosylation of the hydroxylisyl residues. In one embodiment of the present invention, the present invention provides a method for improving the production of proteins, such as hydroxylated protein (e.g., collagen) or carbohydrates in yeast. The method includes inserting (for example, transforming or genomically integrating) a kgtP gene, DNA for a protein or carbohydrate and DNA for promoters, terminators and markers within the yeast to allow the yeast to produce an α-ketoglutarate transporter and the protein or carbohydrate. The method also includes inserting genes that express protein (s) that hydroxyl (m) proline and / or lysine residues.

[008] Another aspect of the invention is directed to modified yeast cells that produce ascorbate. The modification includes the insertion of

Petition 870180132932, of 9/21/2018, p. 12/93 / 80 genes that allow the functioning of an ascorbate synthesis pathway. In one embodiment, the present invention provides a method for producing modified yeast cells that produce ascorbate. The method includes inserting genes necessary to complete a functional ascorbate synthesis pathway. In this method, it is anticipated that it will only be necessary to insert one or more of the genes encoding proteins for the portion of the ascorbate synthesis pathway downstream from the precursor of the ascorbate pathway fed to the yeast cells. In another embodiment, the present invention provides a method for producing intracellular ascorbate in a yeast cell including a medium-modified yeast cell. In another embodiment, the present invention provides a yeast cell modified with a constant amount of intracellular ascorbate.

[009] Yet another aspect of the invention is directed to modified yeast cells that contain an α-ketoglutarate transporter and that produce ascorbate. Yeast cells can be further modified to express and / or over-express a protein that hydroxylates proline and / or lysine residues to yield hydroxyproline, 3-hydroxyproline (Hyp) and hydroxylisin (Hyl) and glycosylation of hydroxylisyl residues. The method includes inserting genes necessary to complete a functional pathway for ascorbate synthesis and an α-ketoglutarate transporter in yeast. The method also includes inserting genes that express protein (s) that hydroxyl (m) proline and / or lysine residues. In another embodiment, the present invention provides a method for producing intracellular ascorbate in a yeast cell including a medium-modified yeast cell. In one embodiment of the present invention, the present invention provides a method for improving the production of proteins such as hydroxylated collagen or carbohydrates in yeast that contain an α-ketoglutarate transporter and that produce ascorbate.

Detailed Description of the Invention

Petition 870180132932, of 9/21/2018, p. 13/93 / 80 [0010] As described and exemplified in this document, yeast cells (for example, Pichia pastoris) can be used to express recombinant bovine collagen Type III with different degrees of hydroxylation. The hydroxylation of the recombinant collagen was achieved by coexpression of bovine P4HA and bovine P4HB that code, respectively, the bovine alpha and beta prolyl-4-hydroxylase subunits, respectively. However, the invention is not limited to Type III collagen products and expression and can be practiced with other proteins or carbohydrates, polynucleotides that encode the subunits of other types of collagen, as well as with enzymes that hydroxyl proline residues, lysine residues or both proline and lysine residues. Type III tropocollagen is a homotrimer. However, in some embodiments, a collagen will form a heterotrimer composed of different polypeptide chains, such as Type I collagen, which is initially composed of two pro-a1 (I) chains and a pro-a2 (I) chain.

[0011] Collagen. Collagen is the main component of leather. Animal skin contains significant amounts of collagen, a fibrous protein. Collagen is a generic term for a family of at least 28 distinct types of collagen; the animal's skin is usually type I collagen, although other types of collagen can be used in the formation of leather, including type III collagen. The term “collagen” encompasses unprocessed (for example, pro-collagen) as well as post-translationally modified and proteolyzed collagens having a triple helical structure. [0012] Collagens are characterized by the repetition of a triplet of amino acids, - (Gly-X-Y) n-, and approximately one third of the amino acid residues in collagens are glycine. X is often proline and Y is often hydroxyproline, although there may be up to 400 possible Gly-XY triplets. Different animals can produce different compositions of collagen amino acids, which can result in different properties and differences in the resulting leather.

Petition 870180132932, of 9/21/2018, p. 14/93 / 80 [0013] The collagen structure can consist of three intertwined peptide chains of different lengths. Triple collagen helices (or monomers) can be produced from alpha chains of about 1,050 amino acids in length, so that the triple helix takes the form of a rod approximately 300 nm long, with a diameter of approximately 1, 5 nm.

[0014] Collagen fibers can have a range of diameters, depending on the type of animal skin. In addition to type I collagen, the skin (leather) can also include other types of collagen, including type III collagen (reticulin), type IV collagen and type VII collagen. There are several types of collagen throughout the mammalian body. For example, in addition to being the main component of skin and animal leather, type I collagen also exists in cartilage, tendon, vascular ligation, organs, muscles and the organic portion of bone. Successful efforts have been made to isolate collagen from various regions of the mammalian body, in addition to the animal's skin or leather. Decades ago, researchers found that, at neutral pH, acid-solubilized collagen self-assembled into fibrils composed of the same cross-patterns that were seen in native tissue; Schmitt F.O. J. Cell. Comp Physiol. 1942; 20: 11). This led to the use of collagen in tissue engineering and a variety of biomedical applications. In more recent years, collagen has been harvested from bacteria and yeasts using recombinant techniques.

[0015] Collagens are formed and stabilized through a combination of physical and chemical interactions, such as electrostatic interactions including salt bridge interactions, hydrogen bonds, Van der Waals, dipole-dipole forces, polarization forces, hydrophobic interactions and covalent bonds, often catalyzed by enzymatic reactions. Several distinct types of collagen have been identified in vertebrates, including bovine, sheep, pig, chicken and

Petition 870180132932, of 9/21/2018, p. 15/93 / 80 humans.

[0016] In general, the types of collagen are numbered by Roman numerals and the chains found in each type of collagen are identified by Arabic numerals. Detailed descriptions of the structure and biological functions of several different types of naturally occurring collagens are available in the art; see, for example, Ayad et al. (1998) The Extracellular Matrix Facts Book, Academic Press, San Diego, CA; Burgeson, R E., and Nimmi (1992) Collagen types: Molecular Structure and Tissue Distribution in Clin. Orthop. 282: 250-272; Kielty, C. M. et al. (1993) The Collagen Family: Structure, Assembly And Organization In The Extracellular Matrix, Connective Tissue And Its Heritable Disorders, Molecular Genetics, And Medical Aspects, Royce, P. M. and B. Steinmann eds., Wiley-Liss, NY, pp. 103-147; and Prockop, D.J- and K.I. Kivirikko (1995) Collages: Molecular Biology, Diseases, and Potentials for Therapy, Annu. Rev. Biochem., 64: 403-434.) [0017] Type I collagen is the main fibrillar collagen of bone and skin, comprising approximately 80-90% of an organism's total collagen. Type I collagen is the main structural macromolecule present in the extracellular matrix of multicellular organisms and comprises approximately 20% of the total protein mass. Type I collagen is a heterotrimeric molecule comprising two α1 (I) and an α2 (I) chains, encoded by the COL1A1 and COL1A2 genes, respectively. In vivo, assembly of Type I collagen fibrils, fibers and bundles of fibers occur during development and provides mechanical support to the tissue while allowing cell mobility and nutrient transport. Other types of collagen are less abundant than type I collagen and exhibit different distribution patterns. For example, type II collagen is the predominant collagen in cartilage and vitreous humor, whereas type III collagen is found at high levels in blood vessels and, to a lesser extent, in

Petition 870180132932, of 9/21/2018, p. 16/93 / 80 skin.

[0018] Type II collagen is a homotrimeric collagen comprising three identical chains of al (II) encoded by the COL2A1 gene. Purified type II collagen can be prepared from tissues by methods known in the art, for example, by procedures described in Miller and Rhodes (1982) Methods In Enzymology 82: 33-64.

[0019] Type III collagen is an important fibrillar collagen found in the skin and vascular tissues. Type III collagen is a homotrimeric collagen comprising three identical a1 (III) chains encoded by the COL3A1 gene. The COL3A1 gene can be optimized for expression in the host cell, for example, Pichia pastoris (SEQ ID NO:

10). Methods for purifying type III collagen from tissues can be found, for example, in Byers et al. (1974) Biochemistry 13: 52435248; and Miller and Rhodes, supra.

[0020] Type IV collagen is found in basal membranes in the form of leaves instead of fibrils. Most commonly, type IV collagen contains two α1 (IV) chains and an α2 (IV) chain. The particular chains comprising type IV collagen are tissue specific. Type IV collagen can be purified using, for example, the procedures described in Furuto and Miller (1987) Methods in Enzymology, 144: 41-61, Academic Press. [0021] Type V collagen is a fibrillar collagen found mainly in bones, tendons, cornea, skin and blood vessels. Type V collagen exists in both homotrimeric and heterotrimeric forms. A form of type V collagen is a heterotrimer with two α1 (V) chains and an α2 (V) chain. Another form of type V collagen is a heterotrimer of the α1 (V), α2 (V) and α3 (V) chains. An additional form of type V collagen is an a1 (V) homotrimer. Methods for isolating type V collagen from natural sources can be found, for example, in Elstow and Weiss (1983) Collagen Rel. Res. 3: 181-193, and Abedin et al. (1982) Biosci.

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Rep. 2: 493-502.

[0022] Type VI collagen has a small triple helical region and two large non-collagenous remaining portions. Type VI collagen is a heterotrimer that comprises the a1 (VI), a2 (VI) and a3 (VI) chains. Type VI collagen is found in many connective tissues. Descriptions of how to purify type VI collagen from natural sources can be found, for example, in Wu et al. (1987) Biochem. J. 248: 373-381, and Kielty et al. (1991) J. Cell Sci. 99: 797-807.

[0023] Type VII collagen is a fibrillar collagen found in certain epithelial tissues. Type VII collagen is a three-chain homotrimeric molecule A1 (VII). Descriptions of how to purify type VII collagen from tissue can be found, for example, in Lunstrum et al. (1986) J. Biol. Chem. 261: 9042-9048 and Bentz et al. (1983) Proc. Natl. Acad. Sci. USA 80: 3168-3172. Collagen type VIII can be found on the Descemet's membrane in the cornea. Type VIII collagen is a heterotrimer that comprises two a1 (VIII) chains and an a2 (VIII) chain, although other chain compositions have been reported. Methods for purifying type VIII collagen from nature can be found, for example, in Benya and Padilla (1986) J. Biol. Chem. 261: 4160-4169, and Kapoor et al. (1986) Biochemistry 25: 3930-3937.

[0024] Type IX collagen is a fibril-associated collagen found in cartilage and vitreous humor. Type IX collagen is a heterotrimeric molecule comprising a1 (IX), a2 (IX) and a3 (IX) chains. Type IX collagen was classified as a FACIT collagen (Collagen associated with fibrillation with interrupted triple helix), having several triple helical domains separated by non-triple helical domains. Procedures for purifying type IX collagen can be found, for example, in Duance, et al. (1984) Biochem. J. 221: 885-889; Ayad et al. (1989) Biochem. J. 262: 753-761; and Grant et al. (1988) The Control of Tissue

Petition 870180132932, of 9/21/2018, p. 18/93 / 80

Damage, Glauert, A. M., ed., Elsevier Science Publishers, Amsterdam, pp. 328.

[0025] Collagen type X is a homotrimeric compound of a1 (X) chains. Type X collagen was isolated from, for example, hypertrophic cartilage found in growth plates; see, for example, Apte et al. (1992) Eur J Biochem 206 (1): 217-24.

[0026] Type XI collagen can be found in cartilaginous tissues associated with type II, type IX collagen and elsewhere in the body. Type XI collagen is a heterotrimeric molecule comprising a1 (XI), a2 (XI) and a3 (XI) chains. Methods for purifying type XI collagen can be found, for example, in Grant et al. supra.

[0027] Type XII collagen is a FACIT collagen found mainly in association with type I collagen. Type XII collagen is a homotrimeric molecule comprising three a1 chains (XII). Methods for purifying type XII collagen and variants thereof can be found, for example, in Dublet et al. (1989) J. Biol. Chem. 264: 13150-13156; Lunstrum et al. (1992) J. Biol. Chem. 267: 20087-20092; and Watt et al. (1992) J. Biol. Chem. 267: 20093-20099. Type XIII is a non-fibrillar collagen found, for example, in the skin, intestine, bone, cartilage and striated muscle. A detailed description of type XIII collagen can be found, for example, in Juvonen et al. (1992) J. Biol. Chem. 267: 24700-24707. [0028] Type XIV is a FACIT collagen characterized as a homotrimeric molecule comprising a1 chains (XIV). Methods for isolating collagen type XIV can be found, for example, in AubertFoucher et al. (1992) J. Biol. Chem. 267: 15759-15764, and Watt et al., Supra. [0029] Type XV collagen is homologous in structure to type collagen

XVIII. Information on the structure and isolation of natural type XV collagen can be found, for example, in Myers et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10144-10148; Huebner et al. (1992) Genomics

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14: 220-224; Kivirikko et al. (1994) J. Biol. Chem. 269: 4773-4779; and Muragaki, J. (1994) Biol. Chem. 264: 4042-4046.

[0030] Type XVI collagen is a fibril-associated collagen, found, for example, in the skin, pulmonary fibroblasts and keratinocytes. Information on the structure of type XVI collagen and the gene encoding type XVI collagen can be found, for example, in Pan et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6565-6569; and Yamaguchi et al. (1992) J. Biochem. 112: 856-863.

[0031] Type XVII collagen is a hemidesmosal transmembrane collagen, also known in bullous pemphigoid antigen. Information on the structure of type XVII collagen and the gene encoding type XVII collagen can be found, for example, in Li et al. (1993) J. Biol. Chem. 268 (12): 8825-8834; and McGrath et al. (1995) Nat. Genet. 11 (1): 83-86.

[0032] Type XVIII collagen is similar in structure to type XV collagen and can be isolated from the liver. Descriptions of the structures and isolation of type XVIII collagen from natural sources can be found, for example, in Rehn and Pihlajaniemi (1994) Proc. Natl. Acad. Sci USA 91: 42344238; Oh et al. (1994) Proc. Natl. Acad. Sci USA 91: 4229-4233; Rehn et al. (1994) J. Biol. Chem. 269: 13924-13935; and Oh et al. (1994) Genomics 19: 494-499.

[0033] Type XIX collagen is believed to be another member of the FACIT collagen family and was found in mRNA isolated from rhabdomyosarcoma cells. Descriptions of the structures and isolation of type XIX collagen can be found, for example, in Inoguchi et al. (1995) J. Biochem. 117: 137-146; Yoshioka et al. (1992) Genomics 13: 884-886; and Myers et al., J. Biol. Chem. 289: 18549-18557 (1994).

[0034] Type XX collagen is a newly found member of the FACIT collagen family and has been identified in the chicken cornea. (See, for

Petition 870180132932, of 9/21/2018, p. 20/93 / 80 example, Gordon et al. (1999) FASEB Journal 13: A1119; and Gordon et al. (1998), IOVS 39: S1128.) [0035] The term collagen refers to any of the known collagen types, including type I to XX collagen described above, as well as any other collagen, whether natural, synthetic, semi-synthetic or recombinant. . All collagens, modified collagens and collagen-like proteins described in this document are included. The term also encompasses collagen and collagen-like proteins or collagen proteins comprising the motif (Gly-X-Y) n in which n is an integer. It includes collagen molecules and collagen-like proteins, trimers of collagen molecules, collagen fibrils and collagen fibril fibers. It also refers to chemically, enzymatically or recombinantly modified collagens or collagen-like molecules that can be fibrillated, as well as collagen fragments, collagen-like molecules and collagen molecules capable of assembling in a nanofiber. Recombinant collagen molecules, whether native or manipulated, will generally comprise the repeated sequence - (Gly-X-Y) not described in this document.

[0036] Collagen in a collagen composition may homogeneously contain a single type of collagen molecule, such as 100% bovine type I collagen or 100% bovine type III collagen, or may contain a mixture of different types of collagen molecules or collagen-like molecules, such as a mixture of bovine Type I and Type III molecules. Such mixtures can include> 0%, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99 or <100% of the individual components of collagen or collagen-like protein. This range includes all intermediate values. For example, a collagen composition can contain 30% type I collagen and 70% type III collagen, or it can contain 33.3% type I collagen, 33.3% type II collagen and 33.3% collagen type III, in which the percentage of

Petition 870180132932, of 9/21/2018, p. 21/93 / 80 collagen is based on the total mass of collagen in the composition or on the molecular percentages of the collagen molecules.

[0037] The "collagen fibrils" are nanofibers composed of tropocolagen (triple helices of collagen molecules). Tropocollagens also include tropocollagen-like structures exhibiting triple helical structures. The collagen fibrils of the invention can have diameters ranging from 1 nm to 1 pm. For example, the collagen fibrils of the invention may have an average or individual fibril diameter of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 nm (1 pm). This range includes all intermediate values. In some embodiments of the invention, collagen fibrils will form networks. Collagen fibrils can associate in fibrils showing a pattern of bands and these fibrils can associate in larger aggregates of fibrils. In some modalities, collagen or collagen-like fibrils will have diameters and orientations similar to those of the top grain or surface layer of a bovine leather or other conventional leather. In other embodiments, collagen fibrils may have diameters comprising the upper grain than those of a corium layer of conventional leather.

[0038] A "collagen fiber" is composed of collagen fibrils that are compacted and exhibit a high degree of alignment in the direction of the fiber. This can vary in diameter from more than 1 pm to more than 10 pm, for example> 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 pm or more. Some embodiments of the collagen fibril network of the invention do not contain a substantial content of collagen fibers with diameters greater than 5 pm. The grain surface composition of a leather may differ from its inner parts, such as corium, which contains bundles of thicker fibers. [0039] Fibrillation refers to a process for producing collagen fibrils. It can be done by raising the pH or adjusting the concentration of

Petition 870180132932, of 9/21/2018, p. 22/93 / 80 salt of a collagen solution or suspension. In the formation of fibrillated collagen, collagen can be incubated to form fibrils for any appropriate period of time, including between 1 min and 24 h and all intermediate values.

[0040] The fibrillated collagen described in this document can, in general, be formed in any shape and / or appropriate thickness, including flat sheets, curved forms / sheets, cylinders, wires and complex forms. These sheets and other shapes can have virtually any linear dimensions including a thickness, width or height greater than 10, 20, 30, 40, 50, 60, 70, 80, 90 mm; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 500, 1,000, 1,500, 2,000 cm or more.

[0041] Fibrillated collagen may not have any substantial amount of higher order structure. In a preferred embodiment, the collagen fibrils will not be bundled and do not form the large collagen fibers found in animal skin and provide a strong and uniform non-anisotropic structure to biofabricated leather.

[0042] In other modalities, some collagen fibrils may be bundled or aligned in structures of a higher order. Collagen fibrils in a biofabricated leather can exhibit an orientation index ranging from 0,> 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0, 8, 0.9, <1.0 or 1.0, where an orientation index of 0 describes collagen fibrils that lack alignment with other fibrils and an orientation index of 1.0 describes collagen fibrils that are completely aligned. This range includes all intermediate values. Those skilled in the art are familiar with the guidance index which is also incorporated by reference to Sizeland, et al., J. Agric. Food Chem. 61: 887-892 (2013) or Basil-Jones, et al., J. Agric. Food Chem. 59: 9972-9979 (2011).

[0043] A biofabricated leather can be fibrillated and processed to contain collagen fibrils that resemble or mimic the properties of

Petition 870180132932, of 9/21/2018, p. 23/93 / 80 collagen fibrils produced by certain species or breeds of animals or by animals raised in particular conditions.

[0044] Alternatively, fibrillation and processing conditions can be selected to provide collagen fibrils other than those found in nature, such as decreasing or increasing the diameter of the fibril, degree of alignment, or degree of crosslinking compared to fibrils in natural leather .

[0045] A network of collagen, sometimes called a hydrogel, can be formed as the collagen is fibrillated or can form a network after fibrillation; in some variations, the collagen fibrillation process also forms a gel-like network. Once formed, the fibrillated collagen network can be further stabilized further by incorporating molecules with di-, tri- or multifunctional reactive groups that include chromium, amines, carboxylic acids, sulfates, sulfites, sulfonates, aldehydes, hydrazides, sulfhydryls, diazolins, aryl-, azides, acrylates, epoxides or phenols. [0046] The fibrillated collagen network can also be polymerized with other agents (for example, polymers that are capable of polymerizing or other suitable fibers), which could be used to further stabilize the matrix and provide the desired final structure. Hydrogels based on acrylamides, acrylic acids and their salts can be prepared using reverse suspension polymerization. The hydrogels described in this document can be prepared from polar monomers. The hydrogels used can be natural polymeric hydrogels, synthetic polymeric hydrogels or a combination of these. The hydrogels used can be obtained using graft polymerization, crosslinking polymerization, networks formed by water soluble polymers, radiation crosslinking and so on. A small amount of crosslinking agent can be added to the hydrogel composition to improve polymerization.

[0047] The average or individual length of the collagen fiber can

Petition 870180132932, of 9/21/2018, p. 24/93 / 80 vary from 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 (1 gm); 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 μιιι (1 mm) across the entire thickness of a biofabricated leather. These ranges include all intermediate values and subintervals.

[0048] Fibrils can align with other fibrils through 50,

100, 200, 300, 400, 500 pm or more of their lengths or may exhibit little or no alignment. In other modalities, some collagen fibrils may be bundled or aligned in structures of a higher order. [0049] Collagen fibrils in a biofabricated leather may exhibit an orientation index ranging from 0,> 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 , 0.8, 0.9, <1.0 or 1.0, where an orientation index of 0 describes collagen fibrils that lack alignment with other fibrils and an orientation index of 1.0 describes collagen fibrils that are completely aligned. This range includes all intermediate values. Those skilled in the art are familiar with the guidance index which is also incorporated by reference to Sizeland, et al., J. Agric. Food Chem. 61: 887-892 (2013) or Basil-Jones, et al., J. Agric. Food Chem. 59: 9972-9979 (2011).

[0050] The density of collagen fibril in a biofabricated leather can vary between about 1 to 1,000 mg / cm ³ , preferably between 5 and 500 mg / cm ³ , including all intermediate values, such as 5, 10, 20, 30 , 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1,000 mg / cm3.

[0051] Collagen fibrils, in a biofabricated leather, can exhibit a unimodal, bimodal, trimodal or multimodal distribution, for example, a biofabricated leather can be composed of two different fibril preparations, each with a different range of diameters of fibrils arranged around one of two different ways. Such mixtures can be selected to impart additive, synergistic or

Petition 870180132932, of 9/21/2018, p. 25/93 / 80 a balance of physical properties in a biofabricated leather conferred by fibrils with different diameters.

[0052] Natural leather products can contain 150 to 300 mg / cm ³ of collagen based on the weight of the leather product. A biofabricated leather may contain a similar content of collagen or collagen fibrils, according to conventional leather, based on the weight of the biofabricated leather, such as a collagen concentration of 100, 150, 200, 250, 300 or 350 mg / cm3.

[0053] Fibrillated collagen, sometimes called a hydrogel, can have a selected thickness based on its final use. The thicker or more concentrated preparations of fibrillated collagen generally produce thicker biofabricated hides. The final thickness of a biofabricated leather can be just 10, 20, 30, 40, 50, 60, 70, 80 or 90% of that of the fibril preparation before shrinkage caused by crosslinking, dehydration and lubrication.

[0054] Cross-linking refers to the formation (or reformation) of chemical bonds within collagen molecules. A cross-linking reaction stabilizes the collagen structure and, in some cases, forms a network between the collagen molecules. Any suitable crosslinking agent known in the art can be used, including, without limitation, mineral salts, such as those based on chromium, formaldehyde, hexamethylene diisocyanate, glutaraldehyde, polyepoxic compounds, gamma irradiation and ultraviolet irradiation with riboflavin. Crosslinking can be carried out by any known method; see, for example, Bailey et al., Radiat. Res. 22: 606-621 (1964); Housley et al., Biochem. Biophys. Commun. 67: 824830 (1975); Siegel, Proc. Natl. Acad. Sci. U.S.A. 71: 4826-4830 (1974); Mechanic et al., Biochem. Biophys. Commun. 45: 644-653 (1971); Mechanic et al., Biochem. Biophys. Commun. 41: 1597-1604 (1970); and Shoshan et al., Biochim. Biophys. Minutes 154: 261-263 (1968), each of the

Petition 870180132932, of 9/21/2018, p. 26/93 / 80 which are incorporated by reference.

[0055] Crosslinkers include isocyanates, carbodiimide, poly (aldehyde), poly (aziridine), mineral salts, poly (epoxies), enzymes, thyrane, phenolics, novolac, resole, as well as other compounds that have chemicals that react with side chains amino acids, such as lysine, arginine, aspartic acid, glutamic acid, hydroxylproline or hydroxylisin.

[0056] A collagen or collagen-like protein can be modified chemically to promote chemical and / or physical cross-linking between the collagen fibers. Chemical cross-linking may be possible because reactive groups, such as the lysine, glutamic acid and hydroxyl groups in the collagen molecule, project from the fibril structure similar to the collagen stem. The crosslinking that involves these groups prevents the collagen molecules from sliding over each other under stress and thus increasing the mechanical strength of the collagen fibers. Examples of chemical cross-linking reactions include, but are not limited to, reactions with the amino group of lysine, or reaction with carboxyl groups on the collagen molecule. Enzymes, such as transglutaminase, can also be used to generate cross-links between glutamic acid and lysine to form a stable γglutamyl-lysine crosslink. The induction of crosslinking between functional groups of neighboring collagen molecules is known in the art. Crosslinking is another step that can be implemented here to adjust the physical properties obtained from materials derived from fibrillated collagen hydrogel. [0057] Collagen still fibrillating or fibrillated can be cross-linked or lubricated. Collagen fibrils can be treated with compounds containing chromium or at least one aldehyde group, or vegetable tannins, before the formation of the network, during the formation of the network or the formation of the network gel. The crosslinking additionally stabilizes the fibrillated collagen leather. For example, collagen fibrils pretreated with acrylic polymer, followed by treatment with a vegetable tannin, such as Acacia Mollissima, can

Petition 870180132932, of 9/21/2018, p. 27/93 / 80 exhibit greater hydrothermal stability. In other embodiments, glyceraldehyde can be used as a crosslinking agent to increase thermal stability, proteolytic resistance and mechanical characteristics, such as Young's modulus between stress of fibrillated collagen tension.

[0058] A biofabricated material containing a network of collagen fibrils may contain 0,> 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20% or more of crosslinking agent , including tanning agents used for conventional leather. The cross-linking agents can be covalently attached to collagen fibrils or other components of a biofabricated material or associated non-covalently with them. Preferably, a biofabricated leather will not contain more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% of a crosslinking agent.

[0059] Lubricating describes a process of applying a lubricant, such as a grease or other hydrophobic compound or any material that modulates or controls the fibril-fibril bond during dehydration, to leather or to biofabricated products comprising collagen. A desirable characteristic of leather aesthetics is the rigidity or handling of the material. In order to achieve this property, the water-mediated hydrogen bond between fibers and / or fibers is limited in leather through the use of lubricants. Examples of lubricants include fats, biological, mineral or synthetic oils, cod oil, sulfonated oil, polymers, organofunctional siloxanes and other hydrophobic agents or compounds used for the greasing of conventional leather, as well as mixtures thereof. Although lubrication is, in some ways, analogous to the greasing of natural leather, a biofabricated product can be treated more uniformly with a lubricant due to its manufacturing method, more homogeneous composition and less complex composition.

[0060] Other lubricants include surfactants, anionic surfactants, cationic surfactants, cationic polymeric surfactants,

Petition 870180132932, of 9/21/2018, p. 28/93 / 80 anionic polymeric surfactants, amphiphilic polymers, fatty acids, modified fatty acids, non-ionic hydrophilic polymers, non-ionic hydrophobic polymers, polyacrylic acids, polymethacrylates, acrylics, natural rubbers, synthetic rubbers, resins, polymers, polymers and copolymers amphiphilic cationic polymers and copolymers and mixtures thereof, as well as emulsions or suspensions thereof in water, alcohol, ketones and other solvents.

[0061] Lubricants can be added to a biofabricated material containing collagen fibrils. Lubricants can be incorporated in any quantity that facilitates the movement of the fibril or that provides leather-like properties, such as flexibility, reduced fragility, durability or water resistance. A lubricant content can vary between 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 , 45, 50, 55, and 60% by weight of biofabricated leather.

[0062] Other additives can be added to modify the properties of leather or biofabricated material. Suitable additives include but are not limited to dyes, pigments, fragrances, resins and microparticles. Resins can be added to modify the elasticity, strength and softness of the material. Suitable resins include, but are not limited to, elastomers, acrylic copolymers, polyurethane and the like. Suitable elastomers include, but are not limited to, styrene, isoprene, butadiene copolymers, such as KRAYTON® elastomers, Hycar® acrylic resins. Resins can be used from about 5% to 200%, or from about 50% to 150% (based on the weight of collagen), these ranges including all values and sub-intervals between 10, 15, 20, 25 , 30, 35, 40, 60, 70, 75, 80, 90, 100, 125, 140 etc.

[0063] Dehydration or water removal describes a process of removing water from a mixture containing collagen fibrils and water, such as an aqueous solution, suspension, gel or hydrogel containing

Petition 870180132932, of 9/21/2018, p. 29/93 / 80 fibrillated collagen. Water can be removed by filtration, evaporation, lyophilization, solvent exchange, vacuum drying, convection drying, heating, irradiation or microwave, or by other known methods for removing water. Additionally, it is known that the chemical crosslinking of collagen removes bound water from collagen by consuming residues of hydrophilic amino acids, such as lysine, arginine and hydroxylisin, among others. The inventors saw that acetone quickly dehydrates collagen fibrils and can also remove water attached to hydrated collagen molecules. The water content of a biofabricated material or leather after dehydration is preferably not more than 60% by weight, for example, not more than 5, 10, 15, 20, 30, 35, 40, 50 or 60% in weight of biofabricated leather. This range includes all intermediate values. The water content is measured by equilibrium at 65% relative humidity at 25 ^o C and 1 atm.

[0064] The grain texture describes a leather-like texture similar, aesthetically or texturally, to the texture of a full grain leather, upper grain leather, corrected grain leather (to which an artificial grain has been applied) or leather texture coarse grain. Advantageously, the biofabricated material of the invention can be adjusted to provide a fine grain, resembling the surface grain of a leather.

[0065] The articles of the invention may include footwear, clothing, gloves, upholstery for furniture or vehicles and other leather articles and products. It includes, but is not limited to, clothes, such as overcoats, jackets, jackets, shirts, pants, shorts, swimsuits, underwear, uniforms, badges or letters, costumes, ties, skirts, dresses, blouses, leggings, gloves, shoe mittens, shoe components, such as sole, tuft, tongue, cuff, rebar, sneakers, sports shoes, running shoes, casual shoes, sports components, runners or casuals, such as toe, toe, outer sole , midsole, upper, laces, eyelets, collar, lining, achilles heel, heel and counter, fashion or shoes

Petition 870180132932, of 9/21/2018, p. 30/93 / 80 women and their shoe components, such as upper outsole, toe cap, toe cap, decoration, vamp, lining, sock, insole, platform, counter, heel or high heel, boots, sandals, buttons, sandals, hats , masks, hats, bandanas, head wraps and belts; jewelry, such as bracelets, watchbands and necklaces; gloves, umbrellas, walking sticks, wallets, cell phone or laptop cases, handbags, backpacks, travel bags, handbags, folios, briefcases, boxes and other personal items; sports, sporting, hunting or recreational equipment, such as harnesses, bridles, reins, wellies, gloves, gloves, tennis rackets, golf clubs, polo, hockey or lacrosse, chess boards and games, medicine balls, cleats, balls baseball and other types of balls and toys; book binding, book covers, frames or works of art; furniture and furnishings for home, office or other interior or exterior, including chairs, sofas, doors, seats, ottomans, partitions, coasters, mouse mats, blotters or other pillows, tables, beds, floor, wall or ceiling , floors; automobiles, boats, aircraft and other vehicular products, including seats, head restraints, upholstery, panels, steering wheels, control levers or covers and other wrappings or covers.

[0066] The physical properties of a biofabricated network of collagen fibrils or biofabricated leather can be selected or adjusted by selecting the type of collagen, the amount of fibrillated collagen concentration, the degree of fibrillation, cross-linking, dehydration and lubrication.

[0067] Many advantageous properties are associated with the network structure of collagen fibrils, which can provide resistant, flexible and substantially uniform properties to the resulting biofabricated material or leather. The preferred physical properties of biofabricated leather according to the invention include a tensile strength ranging from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15 or more MPa, a flexibility determined by elongation at break that varies from 1, 5,

Petition 870180132932, of 9/21/2018, p. 31/93 / 80

10, 15, 20, 25, 30% or more, smoothness, as determined by ISO 17235, 4, 5, 6, 7, 8 mm or more, with a thickness ranging from 0.2, 0.3, 0, 4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3. 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mm or more, and a density of collagen (density of collagen fibrin) of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 200, 300, 400, 500, 600, 700, 800, 900, 1,000 mg / cm ³ or more, preferably 100-500 mg / cm ³ . The ranges above include all sub-ranges and intermediate values.

[0068] Thickness. Depending on its final application, a biofabricated material or leather can have any thickness. Its thickness preferably ranges from about 0.05 mm to 20 mm, as well as any intermediate value within this range, such as 0.05, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 mm or more. The thickness of a biofabricated leather can be controlled by adjusting the collagen content.

[0069] Modulus of elasticity. The elastic modulus (also known as Young's modulus) is a number that measures the resistance of an object or substance to be elastically deformed (that is, not permanently) when a force is applied to it. The modulus of elasticity of an object is defined as the slope of its stress-strain curve in the region of elastic strain. A more rigid material will have a higher modulus of elasticity. The elastic modulus can be measured using a texture analyzer.

[0070] A biofabricated leather can have an elastic modulus of at least 100 kPa. This can range from 100 kPa to 1,000 MPa, as well as any intermediate value in this range, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0, 8, 0.9, 1, 2, 3, 4, 5, 6 7, 8, 9, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 MPa. A biofabricated leather may be able to stretch up to 300% from its relaxed state, for example, by> 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 , 50, 60, 70, 80, 90, 100, 150, 200, 250 or 300%

Petition 870180132932, of 9/21/2018, p. 32/93 / 80 of its relaxed state length.

[0071] Tensile strength (also known as maximum tensile strength) is the ability of a material or structure to support loads tending to stretch, unlike compression resistance, which supports loads that tend to reduce in size. Tensile strength resists tension or tearing, while compressive strength resists compression or pressing.

[0072] A sample of a biofabricated material can be tested for stress resistance using an Instron machine. Clamps are attached to the sample ends and the sample is pulled in opposite directions until failure. Good strength is shown when the sample has a tensile strength of at least 1 MPa. A biofabricated leather can have a tensile strength of at least 1 kPa. This can vary from 1 kPa to 100 MPa, as well as any intermediate value in this range, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 200, 300, 400 500kPA ; 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 MPa.

[0073] Tear strength (also known as tear resistance) is a measure of how well a material can withstand the effects of tearing. More specifically, however, it is how well a material (usually rubber) resists the growth of any cuts when under stress, it is usually measured in kN / m. Tear strength can be measured using the ASTM D 412 method (the same used to measure tensile strength, modulus and elongation). The ASTM D 624 can be used to measure the resistance to the formation of a tear (tear initiation) and the resistance to the expansion of a tear (tear propagation). Regardless of which of these two is being measured, the sample is kept between two insurers and a uniform tensile force is applied until the aforementioned deformation occurs. The tear strength is then calculated by dividing the applied force

Petition 870180132932, of 9/21/2018, p. 33/93 / 80 by the thickness of the material. A biofabricated leather can exhibit tear resistance of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150 or 200% larger than a conventional top grain or other leather of the same thickness comprising the same type of collagen, for example Type I or Type III bovine collagen, processed using the same crosslinker (s) or lubricant ( s). A biofabricated material can have a tear strength ranging from about 1 and 500 N, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500, as well as any intermediate tear resistance within this interval.

[0074] Softness. ISO 17235: 2015 specifies a non-destructive method for determining the softness of leather. It is applicable to all non-rigid leathers, for example shoe leather, upholstery leather, leather of leather goods and leather of clothing. A biofabricated leather can have a smoothness, as determined by ISO 17235, of 2, 3, 4, 5, 6, 7, 8, 10, 11, 12 mm or more.

[0075] Grain. The top grain surface of leather is often considered to be the most desirable due to its smooth texture and smooth surface. The upper grain is a highly porous network of collagen fibrils. The strength and tear resistance of the grain are often a limitation for practical applications of the upper grain alone and conventional leather products are often supported with corium with a much thicker grain. A biofabricated material, as disclosed in this document, which can be produced with strong and uniform physical properties or increased thickness, can be used to supply products similar to superior grains without the need for corium support.

[0076] Content of other components. In some embodiments, collagen is free of other leather components, such as elastin or non-structural animal proteins. However, in some modalities, the content

Petition 870180132932, of 9/21/2018, p. 34/93 / 80 of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, non-collagen structural proteins and / or non-collagen structural proteins in a biofabricated leather can vary from 0, 1, 2, 3 , 4,5, 6, 7, 8, 9 to 10% by weight of biofabricated leather. In other embodiments, a content of actin, keratin, elastin, fibrin, albumin, globulin, mucin, mucinoids, non-collagen structural proteins and / or non-structural collagen proteins can be incorporated into a biofabricated leather in amounts ranging from> 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20% or more by weight of a biofabricated leather . Such components can be introduced during or after fibrillation, crosslinking, dehydration or lubrication.

[0077] Collagen content. The biofabricated material or leather according to the present invention contains an increased collagen content compared to conventional leather. Within the present invention, the collagen content in the biofabricated material or leather varies from 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70% , 75%, 80% or more to 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30% , 25% or less, including all intervals and sub-intervals within the specified lower and upper limits.

[0078] A leather dye refers to dyes that can be used to color leather or biofabricated leather. These include acid dyes, direct dyes, lacquers, sulfur dyes, basic dyes and reactive dyes. Dyes and pigments can also be incorporated into a precursor of a biofabricated leather, such as in a suspension or in a mesh gel comprising collagen fibrils during the production of the biofabricated leather.

[0079] “Fillings”. In some embodiments, a biofabricated leather may comprise padding, in addition to leather components, such as microspheres. One way to control the organization of the

Petition 870180132932, of 9/21/2018, p. 35/93 / 80 network of dehydrated fibrils is to include filling materials that keep the fibers spaced during dehydration. These fillers include nanoparticles, microparticles or various polymers, such as syntans commonly used in the tanning industry. These filling materials can be part of the final dehydrated leather material, or the filling materials can be sacrificial, that is, they are degraded or dissolved leaving an open space for a network of more porous fibrils. The shape and dimension of these fills can also be used to control the orientation of the dehydrated fibril network.

[0080] In some embodiments, a filler may comprise polymeric microsphere (s), granule (s), fiber (s), yarn (s) or organic salt (s). Other materials can also be embedded or otherwise incorporated into a biofabricated leather or a network of collagen fibrils according to the invention. These include, but are not limited to, a fiber, including both woven and non-woven fibers, as well as cotton, wool, cashmere, angora, linen, bamboo, fiber, hemp, soy, marine fibers, fibers produced from milk or milk proteins, silk, spider silk, other peptides or polypeptides including recombinantly produced peptides or polypeptides, chitosan, mycelium, cellulose including bacterial cellulose, wood including wood fibers, rayon, lyocell, vicose, antimicrobial yarn (AMY), Sorbtek, nylon, polyester, elastomers, such as lycra®, spandex or elastane and other polyurethane polyamide polyamides, carbon, including carbon fibers and fullerenes, glass, including glass and non-woven fibers, compounds containing silicon and silicon, minerals, including mineral particles and mineral fibers, and metals or metal alloys, including those which include iron, steel, lead, gold, silver, platinum, copper, zinc and tit uranium, which may be in the form of particles, fibers, threads or other shapes suitable for incorporating into biofabricated leather. Such fills may include material

Petition 870180132932, of 9/21/2018, p. 36/93 / 80 electrically conductive, magnetic material, fluorescent material, bioluminescent material, phosphorescent material or other photoluminescent material or combinations thereof. Mixtures or mixtures of these components can also be embedded or incorporated into a biofabricated leather, for example, to modify the chemical and physical properties described in this document.

[0081] Various forms of collagen are found throughout the animal kingdom. The collagen used in this document can be obtained from animal sources, including vertebrates and invertebrates, or from synthetic sources. Collagen may also come from by-products of existing animal processing. Collagen obtained from animal sources can be isolated using standard laboratory techniques known in the art, for example, Silva et al. Al., Marine Origin Collages and its Potential Applications, Mar. Drugs, 2014 Dec., 12 (12); 5881-5901).

[0082] The collagen described in this document can also be obtained by cell culture techniques including from cells grown in a bioreactor.

[0083] Collagen can also be obtained through techniques of

Recombinant DNA. Constructs encoding non-human collagen can be introduced into yeast to produce non-human collagen. For example, collagen can also be produced with yeast, such as Hansenula polymorpha, Saccharomyces cerevisiae, Pichia pastoris and the like as the host. The recombinant expression of some collagens and collagen-like proteins has been disclosed by Bell, EP 1232182B1, Bovine collagen and method for producing recombinant gelatin; Olsen, et al., U.S. Patent No. 6,428,978, Methods for the production of gelatin and full-length triple helical collagen in recombinant cells; VanHeerde, et al., U.S. Patent No. 8,188,230, Method for Expressing Recombinant Microorganisms and Isolating Collagen-like Polypeptides, Disclosures

Petition 870180132932, of 9/21/2018, p. 37/93 / 80 of which are incorporated herein by reference. However, recombinant collagens were not used to produce leather.

[0084] Materials that are useful in the present invention include, but are not limited to, biofabricated leather materials, natural or synthetic fabrics, non-woven fabrics, knitting fabrics, knitted fabrics and spacer fabrics.

[0085] Any material that retains collagen fibrils can be useful in the present invention. In general, fabrics that are useful have a mesh that ranges from 300 threads per square inch to 1 thread per square foot or a pore size greater than or equal to about 11 pm in diameter. Lace materials can also be useful. In some embodiments, water-soluble fabrics are useful. When used, the portion of the tissue exposed to the collagen solution dissolves to form a void or hole in the tissue and the collagen fills the void or hole. Water-soluble fabrics are typically formed from polyvinyl alcohol fibers and coated with a resin, such as polyvinyl alcohol, polyethylene oxide, hydroxyalkylcellulose, carboxymethylcellulose, polyacrylamide, polyvinylpyrrolidone, polyacrylate and starch. Alternatively, the void or hole may be covered by a secondary material, such as natural or synthetic fabrics, non-woven fabrics, knitted fabrics, knitted fabrics and spacer fabrics.

[0086] Alternatively, the biofabricated leather material can be used to plug a void or hole in the fabric. The size of the void or hole may vary depending on the design to be transmitted. The shape of the void or hole may vary depending on the project. Suitable dimensions of voids or holes can vary from about 0.1 inches to about 5 meters. Suitable formats include, but are not limited to, circles, squares, rectangles, triangles, ellipticals, ovals and brand logos.

[0087] Some materials are used for pre-treatment to improve the

Petition 870180132932, of 9/21/2018, p. 38/93 / 80 connection of biofabricated leather materials. Pretreatment can include collagen coating, resin coating, fabric devouring (also known as the burnout method), chemical or combinations of these. For example, a chemical pretreatment for materials made from cellulose fibers, may include treatment with periodate solution (an oxidizer). Suitable cellulose fabrics are selected from the group consisting of viscose, acetate, lyocell, bamboo and combinations of these. The oxidizer opens sugar rings in the cellulose and allows the collagen to bind to the open rings. The concentration of the oxidant in the solution depends on the extent of oxidation desired. In general, the higher the oxidant concentration or the longer the reaction time, the greater the degree of oxidation achieved. In an embodiment of the present invention, the oxidation reaction can be carried out for a desired period of time to achieve the desired oxidation level. The oxidation reaction can be carried out at various temperatures, depending on the type of oxidizer used. The inventors preferred to use controlled oxidation at room temperature (20 ° C to 25 ° C, preferably 23 ° C) or at room temperature for a period of time from 15 minutes to 24 hours. However, it is also anticipated that temperatures ranging from 17.5 ° C to 30 ° C can also be used. Regarding the time, it is predicted that it is possible for the controlled oxidation to be 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours , 5 hours, 6 hours, 12 hours to 36 hours, 30 hours, 24 hours, 20 hours, 18 hours, 15 hours, including all intervals and sub intervals allowed in this document. The amount of sodium periodate ranges from 25% to 100% by weight of the tissue. As used in this document, offer means the amount of an additive based on the% by weight of cellulose. Other chemical pretreatments are taught in Bioconjugate Techniques by Greg Hermanson, which is incorporated into this document by reference.

Petition 870180132932, of 9/21/2018, p. 39/93 / 80 [0088] The biofabricated leather solution (for example, biofeather), as described in this document, can include any appropriate non-human collagen in addition to and in combination with the recombinant collagen produced by the strains present as discussed in this document.

[0089] As an initial step in the formation of the collagen materials described in this document, the initial collagen material can be placed in solution and fibrillated. The concentration of collagen can vary from approximately 0.5 g / L to 10 g / L, for example, from 0.75 g / L to 8 g / L, from 1 g / L to 7 g / L, from 2 g / L L to 6 g / L, 2.5 g / L to 5 g / L or 3 g / L to 4 g / L. Collagen fibrillation can be induced by introducing salts to the collagen solution. The addition of a salt or a combination of salts, such as sodium phosphate, potassium phosphate, potassium chloride and sodium chloride, to the collagen solution can change the ionic strength of the collagen solution. Collagen fibrillation can occur as a result of increased electrostatic interactions, through increased hydrogen bonding, Van der Waals interactions and covalent bonding. Suitable salt concentrations can range, for example, from approximately 10 mM to 5 M, for example, from 50 mM to 2.5 M, from 100 mM to 1 M or from 250 mM to 500 mM.

[0090] A collagen network can also be highly sensitive to pH. During the fibrillation stage, the pH can be adjusted to control the dimensions of the fibril, such as diameter and length. The appropriate pH can vary from approximately 5.5 to 10, for example, from 6 to 9 or from 7 to 8. After the fibrillation step before filtration, the pH of the solution is adjusted to a pH range of approximately 3, 5 to 10, for example, from 3.5 to 7 or from

3.5 to 5. The overall dimensions and organization of collagen fibrils will affect the stiffness, elasticity and breathability of collagen-derived materials resulting from fibrillation. This can be useful for making leather derived from fibrillated collagen for various uses that may require

Petition 870180132932, of 9/21/2018, p. 40/93 / 80 different stiffness, flexibility and breathability.

[0091] One way to control the organization of the network of dehydrated fibrils is to include filling materials that keep the fibers spaced during drying. Such filling materials can include nanoparticles, microparticles, microspheres, microfibers or various polymers commonly used in the tanning industry. These filling materials can be part of the final dehydrated leather material, or the filling materials can be sacrificial, that is, they are degraded or dissolved leaving an open space for a network of more porous fibrils. [0092] A collagen or collagen-like protein can be modified chemically to promote chemical and / or physical cross-linking between the collagen fibers. Collagen-like proteins were taught in patent application US 2012/0116053 A1, which is incorporated herein by reference. Chemical cross-linking may be possible because the reactive groups, such as the lysine, glutamic acid and hydroxyl groups in the collagen molecule, project from the fibril structure similar to the collagen rod. The crosslinking that involves these groups prevents the collagen molecules from sliding over each other under stress and thus increasing the mechanical strength of the collagen fibers. Examples of chemical cross-linking reactions include, but are not limited to, reactions with the amino group of lysine, or reaction with carboxyl groups on the collagen molecule.

[0093] Enzymes, such as transglutaminase, can also be used to generate cross-links between glutamic acid and lysine to form a stable γ-glutamyl-lysine crosslink. The induction of crosslinking between functional groups of neighboring collagen molecules is known in the art. Crosslinking is another step that can be implemented here to adjust the physical properties obtained from materials derived from fibrillated collagen hydrogel.

Petition 870180132932, of 9/21/2018, p. 41/93 / 80 [0094] Once formed, the fibrillated collagen network can be further stabilized further by incorporating molecules with di-, tri- or multifunctional reactive groups that include chromium, amines, carboxylic acids, sulfates, sulfites, sulfonates, aldehydes, hydrazides, sulfhydryls, diazarines, aryl-, azides, acrylates, epoxides or phenols.

[0095] The fibrillated collagen network can also be polymerized with other agents (for example, polymers that are capable of polymerizing or other suitable fibers) that form a hydrogel or may have fibrous qualities, which could be used to further stabilize the matrix and provide the desired final structure. Hydrogels based on acrylamides, acrylic acids and their salts can be prepared using reverse suspension polymerization. The hydrogels described in this document can be prepared from polar monomers. The hydrogels used can be natural polymeric hydrogels, synthetic polymeric hydrogels or a combination of these. The hydrogels used can be obtained using graft polymerization, crosslinking polymerization, networks formed by water soluble polymers, radiation crosslinking and so on. A small amount of crosslinking agent can be added to the hydrogel composition to improve polymerization.

[0096] The viscosity of the collagen solution can vary from 1 cP to 1000 cP at 20 ° C, including all values and intervals between them, such as 10, 20, 30, 50, 75, 90, 100, 150 , 225, 300, 400, 450, 500, 525, 575, 600, 650, 700, 800, 900 etc. Solutions can be poured, sprayed, painted or applied to a surface. Viscosity can vary depending on how the final material is formed. When a higher viscosity is desired, known thickening agents, such as carboxymethylcellulose and the like, can be added to the solution. Alternatively, the amount of collagen in the solution can be adjusted to vary the viscosity.

Petition 870180132932, of 9/21/2018, p. 42/93 / 80 [0097] The flexibility in the collagen solution allows the production of new materials made entirely through the deposition of the said collagen solution, for example, the creation of biofabricated leather lace materials or three-dimensional materials. In a way, the collagen solution can function as a liquid leather to form, for example, biofabricated leather or bi-leather. The liquid leather can be poured, pipetted, sprayed through a nozzle or applied robotically or a secondary material can be immersed in the liquid leather. A textured surface can be obtained by using a material with holes in the material formation process. Liquid leather also allows the use of masking, stamping and molding techniques. The application of the biofabricated leather solution also allows to modify the properties of the material to which it is applied. For example, the biofabricated leather solution can make the final material stronger, more malleable, more rigid, more flexible, more elastic or soft.

[0098] As mentioned, a biofabricated leather material derived from the methods described above can have thick structural and physical characteristics similar to leather produced from animal skins. In general, in addition to the collagen produced by the present yeast cells, the biofabricated leather materials described in this document can be derived from sources other than leaves or pieces of animal skin, although animal skin may be the source of the collagen used to prepare fibrillated collagen. The source of collagen or collagen-like proteins can be isolated from any animal source (e.g., mammal, fish) or, more particularly, cultured from cell / tissue (including, particularly, microorganisms).

[0099] The biofabricated leather material may include agents that stabilize the fibril network contained therein or may contain agents that promote fibrillation. As mentioned in the previous sections, agents

Petition 870180132932, of 9/21/2018, p. 43/93 / 80 crosslinking (to provide additional stability), nucleating agents (to promote fibrillation) and additional polymerization agents (for additional stability) can be added to the collagen solution before fibrillation (or after) to obtain a fibrillated collagen material with desired characteristics (for example, strength, fold, elasticity and so on).

[00100] As mentioned, after dehydration or drying, the manipulated biofabricated leather materials derived from the methods discussed above have a water content of 20% or less by weight, 17.5% or less by weight, 15% or less by weight, 12.5% or less by weight or 10% or less by weight. The water content of the manipulated biofabricated leather materials can be improved in the finishing steps to obtain leather materials with different purposes and desired characteristics. [00101] As mentioned, any of these biofabricated leathers can be tanned (for example, using a tanning agent including vegetables (tannins), chromium, alum, zirconium, titanium, iron salts or a combination thereof, or any other appropriate tanning). Thus, in any of the resulting biofabricated leather materials described in this document, the resulting material may include a percentage (for example, between 0.01% and 10%, between 0.025% and 8%, between 0.05% and 6%, between 0.1% and 5%, between 0.25% and 4%, between 0.5% and 3%, between 0.75% and 2.5% or between 1% and 2%) of a residual tanning agent (eg tannin, chromium, etc.). Thus, the collagen fibrils in the resulting biofabricated leather material are modified to be tanned, for example, cross-linked to resist degradation.

[00102] Biofabricated leather materials can be treated to provide surface textures. Adequate treatments include, but are not limited to, embossing, debossing and vacuum forming with a plate with holes below the material.

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As is known in the art, the pressure and temperature at which low and high relief engravings are made can vary depending on the desired texture and design. Surface coatings and finishes known in the leather industry can be applied to biofabricated leather materials.

[00103] As mentioned above, in any of the variations to make the biofabricated leathers described in this document, the material can be tanned (cross-linked) as the collagen is fibrillated and / or separately after the occurrence of fibrillation, before dehydration. For example, tanning can include crosslinking using an aldehyde (for example, Relugan GTW) and / or any other tanning agent. Thus, in general, a tanning agent includes any collagen fibril crosslinking agent, such as aldehydes, crosslinkers, chromium, amine, carboxylic acid, sulfate, sulfite, sulfonate, aldehyde, hydrazide, sulfhydryl and diazirine.

[00104] Some methods for making a material including a biofabricated leather material include providing a material, pre-treating the material to make it suitable for bonding with collagen, applying the collagen solution to the material and drying. Drying can include vacuum removal of water, heated air drying, room air drying, heated pressing and pressure drying. When pretreatment is necessary, pretreatment is to cut voids or holes in the material, chemically remove certain fibers and treat the material with a chemical or collagen solution. Other methods do not require pre-treatment of the material. When pretreatment is not necessary, the material is partially soluble in water or retains collagen, but allows water to pass through. Suitable mesh sizes range from 300 lines per square inch to 1 line per square foot. As used in this document, the term bonded or bonded to the fabric means attached so that the biofabricated leather does not peel off easily from the fabric when pulled by hand. A suitable method for testing

Petition 870180132932, of 9/21/2018, p. 45/93 / 80 the effectiveness of the bond is a peel strength test performed on an instrument, such as the Instron® material testing machine. The claws of the machine are attached to the biofabricated leather material and the material to which it was glued, and the claws are separated until the materials tear or separate. The tear strength is reported in N / mm. Suitable peeling forces range from about 0.5 N / mm N / cm ² to 100 N / mm, including 0.75 N / mm to 75 N / mm, or from 1 N / mm to 50 N / mm, from 1.5 N / mm to 25 N / mm, including all intervals or subintervals defined by the above and below limits.

[00105] Hydroxylation of proline and lysine residues into a protein (eg, collagen). The main post-institutional modifications of protein polypeptides containing proline and lysine, including collagen, are the hydroxylation of proline and lysine residues to yield 4-hydroxyproline, 3-hydroxyproline (Hyp) and hydroxylisin (Hyl), and glycosylation of hydroxylisyl residues. These modifications are catalyzed by three hydroxylases - prolyl 4-hydroxylase, prolyl-3-hydroxylase and lysyl hydroxylase - and two glycosyl transferases. In Vivo, these reactions occur until the polypeptides form the triple helical collagen structure, which inhibits further modifications.

[00106] Prolyl-4-hydroxylase. This enzyme catalyzes the hydroxylation of proline residues to (2S, 4R) -4-hydroxyproline (Hyp). Gorres, et al., Critical Reviews in Biochemistry and Molecular Biology 45 (2): (2010) which is incorporated by reference. The Examples below employ tetrameric bovine prolyl-4 hydroxylase (2 alpha and 2 beta chains) encoded by P4HA (SEQ ID NO: 8 (optimized for Pichia ') or SEQ ID NO: 15 (native)) and P4HB (SEQ ID NO: 9 (optimized for Pichia) or SEQ ID NO: 16 (native)), however, isoforms, orthologs, variants, fragments and prolyl-4-hydroxylase from non-bovine sources (eg human prolyl-4-hydroxylase) can also be used since they retain hydroxylase activity in a

Petition 870180132932, of 9/21/2018, p. 46/93 / 80 yeast host cell. Another example of P4HA1 is further described by http://www.omim.org/entry/176710 (Cytogenetic location: 10q22.1; Genomic coordinates (GRCh38): 10: 73,007,21673,096,973 (from NCBI)) and another example of P4HB1 is further described by http://www.omim.org/entry/176790 (Cytogenetic location: 17q25.3; Genomic Coordinates (GRCh38): 17: 81,843,15781,860,667 (from NCBI)) both which are incorporated by reference.

[00107] In the context of the present application, a "variant" includes an amino acid sequence with at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97 , 5%, 98% or 99% sequence identity or similarity to a reference amino acid, such as a sequence of amino acids P4HA and P4HB, using a similarity matrix, such as BLOSUM45, BLOSUM62 or BLOSUM80, in which BLOSUM45 can be used for closely related sequences, BLOSUM62 for mid-range sequences and BLOSUM80 for more distantly related sequences. Except where indicated, a similarity score will be based on the use of BLOSUM62. When using BLASTP, the percentage similarity is based on the positive BLASTP score and the percentage string identity is based on the BLASTP identity score. BLASTP identities show the number and fraction of total residues in pairs of high-score strings that are identical; and BLASTP Positives shows the number and fraction of residues for which the alignment scores have positive values and are similar to each other. Amino acid sequences with these degrees of identity or similarity or any intermediate degree of identity or similarity with the amino acid sequences disclosed in this document are contemplated and covered by this disclosure. A representative BLASTP configuration that uses an Expect Threshold of 10, a Word Size of 3, BLOSUM 62 as

Petition 870180132932, of 9/21/2018, p. 47/93 / 80 a matrix and Gap Penalty of 11 (Existence) and 1 (Extension) and a matrix adjustment of conditional composition score. Other standard BLASTP configurations are described and incorporated by reference to the disclosure available at:

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE= BlastSearch & LINK_LOC = blasthome (last accessed 14 August 2017). Within the present invention, the "variant" retains prolyl4-hydroxylase activity.

[00108] In one embodiment of the present invention, yeast cells are manipulated to produce excess prolyl-4-hydroxylase. A non-limiting example is the incorporation of a polynucleotide that encodes prolyl-4-hydroxylase, an isoform of this, an ortholog of this, a variant of this or a fragment thereof that expresses the activity of prolyl-4 hydroxylase in an expression vector. In one embodiment of the present invention, the expression vector containing the polynucleotide encoding prolyl-4-hydroxylase, an isoform thereof, an ortholog thereof, a variant thereof or a fragment thereof which expresses the activity of prolyl-4-hydroxylase is under the control of an inducible promoter. Suitable yeast cells, expression vectors and promoters are described below.

[00109] Prolyl-3-hydroxylase. This enzyme catalyzes the hydroxylation of proline residues. Precursor of 3-hydroxylase 1 [Bos taurus] is described by the NCBI Reference Sequence NP_001096761.1 (SEQ ID NO: 4) or by NM_001103291.1 (SEQ ID NO: 5). For further description, see Vranka, et al., J. Biol. Chem. 279: 23615-23621 (2004) or http://_www.omim.org/entry/610339 (last accessed on July 14, 2017) which is incorporated by reference. This enzyme can be used in its native form. However, isoforms, orthologs, variants, fragments and prolyl-3-hydroxylase from non-bovine sources can also be used as long as they maintain hydroxylase activity in a host cell of

Petition 870180132932, of 9/21/2018, p. 48/93 / 80 yeast.

[00110] In the context of the present application, a "variant" includes an amino acid sequence with at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97 , 5%, 98% or 99% sequence identity or similarity to a reference amino acid, such as a prolyl-3-hydroxylase amino acid sequence, using a similarity matrix, such as BLOSUM45, BLOSUM62 or BLOSUM80, in which BLOSUM45 can be used for closely related sequences, BLOSUM62 for midrange sequences and BLOSUM80 for more distantly related sequences. Except where indicated, a similarity score will be based on the use of BLOSUM62. When using BLASTP, the percentage similarity is based on the positive BLASTP score and the percentage string identity is based on the BLASTP identity score. BLASTP identities show the number and fraction of total residues in pairs of high-score strings that are identical; and BLASTP Positives shows the number and fraction of residues for which the alignment scores have positive values and are similar to each other. Amino acid sequences with these degrees of identity or similarity or any intermediate degree of identity or similarity with the amino acid sequences disclosed in this document are contemplated and covered by this disclosure. A representative BLASTP configuration that uses an Expect Threshold of 10, a Word Size of 3, BLOSUM 62 as a matrix and Gap Penalty of 11 (Existence) and 1 (Extension) and a conditional composition score matrix adjustment. Other standard BLASTP configurations are described and incorporated by reference to the disclosure available at:

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE= BlastSearch & LINK_LOC = blasthome (last accessed 14 August 2017). Within the present invention, the “variant” retains the activity of prolilPetição 870180132932, of 9/21/2018, p. 49/93 / 80

4-hydroxylase.

[00111] In one embodiment of the present invention, yeast cells are engineered to overproduce prolyl-3-hydroxylase. A non-limiting example is the incorporation of a polynucleotide that encodes prolyl-3-hydroxylase, an isoform of this, an ortholog of this, a variant of this or a fragment thereof that expresses the activity of prolyl-3 hydroxylase in an expression vector. In one embodiment of the present invention, the expression vector containing the polynucleotide encoding prolyl-3-hydroxylase, an isoform of this, an ortholog of this, a variant of this or a fragment thereof that expresses the activity of prolyl-3-hydroxylase is under the control of an inducible promoter. Suitable yeast cells, expression vectors and promoters are described below.

[00112] Lysyl hydroxylase. Lysyl hydroxylase (EC 1.14.11.4) catalyzes the formation of hydroxylisin by hydroxylating lysine residues in X-lys-gly sequences. The enzyme is a homodimer that consists of subunits with a molecular mass of about 85 kD. No significant homology was found between the primary hydroxylase structures and the 2 types of prolyl-4-hydroxylase subunits (176710, 176790), despite the striking similarities in the kinetic properties between these 2 collagen hydroxylases. The hydroxylisin residues formed in the lysyl hydroxylase reaction, for example, in collagen, have 2 important functions: first, their hydroxyl groups serve as binding sites for carbohydrate units, the monosaccharide galactose or the disaccharide glucosylgalactose; and second, they stabilize the collagen intermolecular cross-links.

[00113] An exemplary PLOD1 lysyl hydroxylase pro-collagen-lysine, 2oxoglutarate 5-dioxigenase 1 [Bos taurus (bovine)] is described by Gene ID: 281409 (SEQ ID NO: 6), updated on May 25, 2017 and incorporated by reference to https://www.ncbi.nlm.nih.gov/gene/281409 (accessed by

Petition 870180132932, of 9/21/2018, p. 50/93 / 80 last time on July 14, 2017). Another example is described by SEQ ID NO: 7, which describes Bos taurus lysyl oxidase (LOX). Yet another example is described in SEQ ID NO: 14, which is a bifunctional Bos taurus arginine demethylase and lysyl hydroxylase JMJD6. The enzyme lysyl hydroxylase can be used in its native form. However, isoforms, orthologs, variants, fragments and lysyl hydroxylase from non-bovine sources can also be used as long as they maintain hydroxylase activity in a yeast host cell.

[00114] In the context of the present application, a "variant" includes an amino acid sequence with at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97 , 5%, 98% or 99% sequence identity or similarity to a reference amino acid, such as a lysyl hydroxylase amino acid sequence, using a similarity matrix, such as BLOSUM45, BLOSUM62 or BLOSUM80, in which BLOSUM45 can be used for closely related sequences, BLOSUM62 for mid-range sequences and BLOSUM80 for more distantly related sequences. Except where indicated, a similarity score will be based on the use of BLOSUM62. When using BLASTP, the percentage similarity is based on the positive BLASTP score and the percentage string identity is based on the BLASTP identity score. BLASTP identities show the number and fraction of total residues in pairs of high-score strings that are identical; and BLASTP Positives shows the number and fraction of residues for which the alignment scores have positive values and are similar to each other. Amino acid sequences with these degrees of identity or similarity or any intermediate degree of identity or similarity with the amino acid sequences disclosed in this document are contemplated and covered by this disclosure. A representative BLASTP configuration that uses an Expect Threshold of 10, a Word Size of 3, BLOSUM 62 as

Petition 870180132932, of 9/21/2018, p. 51/93 / 80 a matrix and Gap Penalty of 11 (Existence) and 1 (Extension) and a matrix adjustment of conditional composition score. Other standard BLASTP configurations are described and incorporated by reference to the disclosure available at:

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE= BlastSearch & LINK_LOC = blasthome (last accessed 14 August 2017). Within the present invention, the "variant" retains prolyl4-hydroxylase activity.

[00115] In one embodiment of the present invention, yeast cells are manipulated to produce excess lysyl hydroxylase. A non-limiting example is the incorporation of a polynucleotide that encodes lysyl hydroxylase, an isoform of this, an ortholog of this, a variant of this or a fragment thereof that expresses the activity of lysyl hydroxylase in an expression vector. In one embodiment of the present invention, the expression vector containing the polynucleotide encoding the lysyl hydroxylase, an isoform thereof, an ortholog thereof, a variant thereof or a fragment thereof expressing lysyl hydroxylase activity is under the control of a inducible promoter. Suitable yeast cells, expression vectors and promoters are described below.

[00116] α-ketoglutarate transporter. The kgtP gene exists in Escherichia coli and encodes an α-ketoglutarate transporter that is responsible for the absorption of α-ketoglutarate through the boundary membrane and the concomitant import of a cation (Membrane Topology Model of Escherichia coli oL-Ketoglutarate Permease by PhoA Fusion Analysis , WONGI SEOLt AND AARON J. SHATKIN, JOURNAL OF BACTERIOLOGY, Jan. 1993, p. 565-567). The gene was transformed into cyanobacteria (Uptake of 2-Oxoglutarate in Synechococcus Strains Transformed with the Escherichia coli kgtP Gene, MARI A FE LIX VA 'ZQUEZ-BERMU' DEZ, ANTONIA HERRERO, AND ENRIQUE FLORES,

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JOURNAL OF BACTERIOLOGY, Jan. 2000, p. 211-215).

[00117] The inventors found that turning the kgtP gene into yeast provides a healthier organism. In addition, if the yeast produces a protein, such as collagen or carbohydrate, the transformed yeast increases the production of protein or carbohydrate and improves collagen hydroxylation, creating more α-ketoglutarate available in the endoplasmic reticulum, in which proteins or carbohydrates are made. and hydroxylation occurs.

[00118] An example of a kgtP gene is established in SEQ ID NO: 2, whereas an exemplary α-ketoglutarate transporter is established in SEQ ID NO: 1. This enzyme can be used in its native form. However, isoforms, orthologs, variants, fragments and transporters of α-ketoglutarate from sources other than E.coli can also be used as they maintain α-ketoglutarate transport activity in a yeast host cell. In addition, the kgtP gene can be optimized for expression in yeast. For example, an example of an optimized α-ketoglutarate transporter (kgtP) gene for expression in Pichia is set out in SEQ ID NO: 3.

[00119] In the context of the present application, a "variant" includes an amino acid sequence with at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97 , 5%, 98% or 99% sequence identity or similarity to a reference amino acid, such as an α-ketoglutarate transporter amino acid sequence, using a similarity matrix, such as BLOSUM45, BLOSUM62 or BLOSUM80, in which BLOSUM45 can be used for closely related sequences, BLOSUM62 for mid-range sequences and BLOSUM80 for more distantly related sequences. Except where indicated, a similarity score will be based on the use of BLOSUM62. When using BLASTP, the percentage similarity is based on the positive score of

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BLASTP and the percentage string identity is based on the BLASTP identity score. BLASTP identities show the number and fraction of total residues in pairs of high-score strings that are identical; and BLASTP Positives shows the number and fraction of residues for which the alignment scores have positive values and are similar to each other. Amino acid sequences with these degrees of identity or similarity or any intermediate degree of identity or similarity with the amino acid sequences disclosed in this document are contemplated and covered by this disclosure. A representative BLASTP configuration that uses an Expect Threshold of 10, a Word Size of 3, BLOSUM 62 as a matrix and Gap Penalty of 11 (Existence) and 1 (Extension) and a conditional composition score matrix adjustment. Other standard BLASTP configurations are described and incorporated by reference to the disclosure available at:

https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE= BlastSearch & LINK_LOC = blasthome (last accessed 14 August 2017). Within the present invention, the "variant" retains prolyl4-hydroxylase activity.

[00120] In one embodiment of the present invention, yeast cells are manipulated to overproduce the α-ketoglutarate transporter. A non-limiting example is the incorporation of a polynucleotide that encodes the α-ketoglutarate transporter, an isoform of this, an ortholog of this, a variant of this or a fragment thereof that expresses the activity of the α-ketoglutarate transporter in an expression vector. In one embodiment of the present invention, the expression vector containing the polynucleotide encoding the α-ketoglutarate transporter, an isoform thereof, an ortholog of this, a variant thereof or a fragment thereof expressing the activity of the α-ketoglutarate transporter is under the control of an inducible promoter. Suitable yeast cells, vectors of

Petition 870180132932, of 9/21/2018, p. 54/93 / 80 expression and promoters are described below.

[00121] Polynucleotide (s) encoding one or more polypeptides that allow an ascorbate synthesis pathway to work [00122] L-ascorbic acid, the ascorbate biosynthetic pathway in yeast, plants and animals is described, for example, in Smirnoff (2001) Vitamins & Hormones 61-241-266 and Vitamin C. Functions and biochemistry in animals and plants Asard et al (ed.). Garland Science / BIOS Scientific Publishers, 2004. Several genes, individually or in combination, can be used to allow ascorbate synthesis in yeast. These include, for example, for a plant pathway, the genes or polynucleotides that encode GDP-L-Gal phosphorylase, which converts GDP-L-Galactose into LGalactose-1-P, Inositol-phosphate phosphatase which converts L- Galactose-1-P to L-galactose, GDP-mannose-3,5-epimerase, which converts GDP-D-mannose to GDP-L-galactose, can be transformed into yeast. Pichia already has the other genes needed to complete the path. These genes provide the following enzymes: hexokinase, Glucose-6-phosphate-isomerase, Mannose-6-phosphate-isomerase, Phosphomanomutase and Mannose-1-phosphate-guanylyltransferase. [00123] For an animal route, the following genes can be added: L-gulono-1,4-lactone oxidase, which converts L-Gulon-1,4-lactone to L-Ascorbate, aldonolactonase, which converts L-gluconic acid for LGulono-1,4-lactone, glucuron lactone reductase, which converts Dglucuronic acid to D-glucuron lactone, D-glucuronate reductase, which converts D-glucuronic acid to L-gluconic acid, uronolactonase which converts D-Glucuronic acid to D- Glucuron lactone, D-glucuron kinase, which converts D-glucuronic acid-1-P to D-glucuronic acid, glucuronate-1 phosphate uridylyltransferase, which converts UDP-Glucuronic acid to D-Glucuronic acid-1-P, UDP-D-glucose dehydrogenase, which converts UDP-DGglucose to UDP-Glucuronic acid, UTP-glucose-1-phosphate uridylyltransferase, which converts D-Glucose-1-P to UDP-D-Glucose,

Petition 870180132932, of 9/21/2018, p. 55/93 / 80 phosphoglucomutase, which converts D-Glc-6-P to D-Glucose-1-P and / or hexokinase, which converts D-Glucose to D-Glc-6-P.

[00124] It is not necessary to transform all genes for all enzymes to allow the yeast to ascorbate. In fact, in the present invention, it is anticipated that it will be necessary to insert only one or more of the genes encoding proteins for the portion of the ascorbate synthesis pathway downstream from the precursor of the ascorbate pathway fed to the yeast cells. For example, it is possible to insert the gene for the enzyme Inositol-phosphate phosphatase which converts L-Galactose-1-P to L-Galactose in yeast and then feeds L-Galactose-1-P in yeast, which allows the yeast to produce L-ascorbate.

[00125] The phosphorylase of GDP-L-Gal can be supplied from Arabidopsis thaliana and includes all or part of the nucleotide sequence of SEQ ID NO: 21.

[00126] A representative amino acid sequence of GDP-L-Gal phosphorylase is as defined in SEQ ID NO: 22.

[00127] Inositol phosphate phosphatase (EC 3.1.3.25) is a known class of enzymes and is a phosphatase acting on L-galactose 1-phosphate (L-Gal 1-P), D-myoinositol 3-phosphate (D- Ins 3-P) and D-myoinositol 1-phosphate (D-Ins 1P). As substrates, it is also possible to use beta-glycerophosphate (glycerol 2-P) and, to a lesser extent, D-galactose 1-phosphate (D-Gal 1-P), alpha-D-glucose 1-phosphate (aD-Glc 1 -P), D-mannitol 1-phosphate and adenosine 2'-monophosphate. No activity with D-fructose 1-phosphate (D-Fru 1-P), fructose 1,6-bisphosphate (Fru 1,6-bisP), D-glucose 6-phosphate (D-Glc 6-P), D- alpha-glycerophosphate (glycerol 3P), D-sorbitol 6-phosphate and D-myoinositol 2-phosphate. The position of C1 phosphate on a six-membered ring substrate is important for catalysis. The amino acid positions of Arabidopsis thaliana at 71, 91, 93, 94 and 221 contribute to metal binding and amino acid positions 71 and 213 contribute to substrate binding. A nucleotide sequence

Petition 870180132932, of 9/21/2018, p. Representative 56/93 / 80 is shown in SEQ ID NO: 23.

[00128] An Inositol-phosphate phosphatase isoform is shown in SEQ ID NO: 24.

[00129] GDP-mannose-3,5-epimerase (EC 5.1.3.18) is a known class of enzymes and catalyzes a reversible epimerization of GDP-D-mannose that precedes the step committed in the biosynthesis of vitamin C (L-ascorbate ), resulting in the hydrolysis of the highly energetic glycosyl-pyrophosphoryl bond. It is capable of catalyzing 2 distinct epimerization reactions and can release both GDP-L-galactose and GDP-L-gulose from GDP-mannose. In Arabidopsis thaliana, regions 143-145, 216-21 and / or 241-243 are involved in substrate binding with one or more positions 145, 174, 178, 217 and / or 306 involved in enzymatic activity and one or more positions 58, 78, 174 and / or 178 involved in NAD binding, one or more of positions 103, 203, 225, 306 and / or 356 involved in substrate binding.

[00130] The polynucleotide used in this document can encode all or part of the amino acid sequence of SEQ ID NO: 25.

[00131] L-gulono-1,4-lactone oxidase (CE 1.1.3.8) is a known class of enzymes and is involved in ascorbic acid biosynthesis. In Arabidopsis thaliana, position 156 is involved in activity and preferably contains only amino acids 102 to 610 or a fragment of these with amino acids 123-258 involved in the binding of FAD. [00132] The polynucleotide used in this document can encode all or part of the amino acid sequence of at least one isoform, for example, SEQ ID NO: 26.

[00133] A second isoform from the above sequence in 494512 in which the sequence is changed from KSPISPAFSTSEDDIFSWV (SEQ ID NO: 27) and WYNHVPPDSRPSPEKGHHR (SEQ ID NO: 28) and does not contain 513-610.

[00134] Glucuron lactone reductase (CE 1.1.1.20) is a class

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49/80 known enzymes and catalyzes a reaction of L-gulono-1,4-lactone + NADP (+) <=> D-glucurono-3,6-lactone + NADPH and is known in the art, for example, from from a number of organizations, as highlighted below, from the publicly accessible UniProt database:

UNIPROT ENTRY NAME ORGANISMS N ° OF AA MOLECULAR WEIGHT [Da] Q3KFB7 pBLAST Q3KFB7_PSEPF Pseudomonas fluorescens (strain PfD-1) 797 87565 B4ECW4 pBLAST B4ECW4BURCJ Burkhokleria cenocepacia (strain ATCC BAA-245 / DSM 16553 / LMG 16656 / NCTC 13227 / J2315 / CF5610) 533 57165 J7QMF7 pBLAST J7QMF7ECOLX Escherichia coli 229 24313 G8Q002 pBLAST G8Q002 PSEFL Pseudomonas fluorescens F113 799 87653 QOJZZ6 pBLAST Q0JZZ6_CUPNH Cupriavidus necator (strain ATCC 17699 / H16 / DSM 428 / Stanier 337) 177 18548 Q63RE5 pBLAST Q63RE5_BURPS Burkholderia pseudomallei (strain K96243) 787 84441 Q3JPBG pBLAST Q3JPB6BURP1 Burkholderia pseudomallei (estirpel710b) 505 54737 B4ECW3 pBLAST B4ECW3BURCJ Burkhokleria cenocepacia (strain ATCC BAA-245 / DSM 16553 / LMG 16656 / NCTC 13227 / J2315 Z CF5610) 787 84879 C3KN28 pBLAST C3KN28_SINFN Sinortiizobium fredií (strain NBRC 101917 / NGR234) 117 12377 J7RQ89 pBLAST J7RQ89ECOLX Escherichia coli chi7122 229 24313 Q63RE4 pBLAST Q63RE4_BURPS Burkholderia pseudomallei (strain K96243) 505 54737 C9YHB1 pBLAST C9YHB1_9BURK Curvibacter putative symbiont of Hydra magn ipapillata 755 81539 Q3JPB7 pBLAST Q3JPB7BURP1 Burkholderia pseudomallei (strain 1710o) 787 84441 C3JZH5 pBLAST C3JZH5PSEFS Pseudomonas fluorescens (strain SBW25) 799 87677

[00135] Aldonolactonase is known in the art and, in some cases, is also called a gluconolactonase (EC 3.1.1.17) and is known from several organisms, as outlined below, from the publicly accessible UniProt database:

Input Input name Gene names Body Length G8Q002 G8Q002_PSEFL PSF113_4031 Pseudomonas fluorescens F113 799 J7QMF7 J7QMF7_ECOLX yagT cutS, AA102_15875, ACN002_0308, ACN77_07560, ACN81 05350 Escherichia coli 229 Q63RE4 Q63RE4_BURPS xdhA BPSL2728 Burkholderia pseudomallei (strain K96243) 505 B4ECW4 B4ECW4_BURCJ xdhA BCAL3173 Burkholderia cenocepacia (ATCC strain BAA-245 / DSM 16553 / LMG 16656 / NCTC 13227 / J2315 / CF5610) (Burkholderia cepacia (strain J2315)) 533

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Input Input name Gene names Body Length Q3JPB6 Q3JPB6_BURP1 xdhA BURPS1710b 3215 Burkholderia pseudomallei (strain 1710b) 505 C9YHB1 C9YHB1_9BURK XDH Csp_B21610 Curvibacter putative symbiont of Hydra magnipapillata 755 Q0JZZ6 Q0JZZ6_CUPNH xdhC2 H16_B1896 Cupriavidus necator (ATCC strain 17699 / H16 / DSM 428 / Stanier 337) (Ralstonia eutropha) 177 C3KN28 C3KN28_SINFN yagT NGR_b01350 Sinorhizobium fredii (strain NBRC 101917 / NGR234) 117 J7RQ89 J7RQ89 ECOLX yagT BN16 07581 Escherichia coli chi7122 229 Q63RE5 Q63RE5_BURPS xdhB BPSL2727 Burkholderia pseudomallei (strain K96243) 787 B4ECW3 B4ECW3_BURCJ xdhB BCAL3172 Burkholderia cenocepacia (ATCC strain BAA-245 / DSM 16553 / LMG 16656 / NCTC 13227 / J2315 / CF5610) (Burkholderia cepacia (strain J2315)) 787 Q3JPB7 Q3JPB7_BURP1 xdhB BURPS1710b 3213 Burkholderia pseudomallei (strain 1710b) 787 Q3KFB7 Q3KFB7_PSEPF Pfl01_1796 Pseudomonas fluorescens (strain Pf0-1) 797 C3JZH5 C3JZH5_PSEFS PFLU_4593 Pseudomonas fluorescens (strain SBW25) 799 A0A1W1 B2Y5 A0A1W1B2Y5_9B URK xdhA UA11_02398, UA12 02333 Burkholderia multivorans 531 A0A1W0Z 2H1 A0A1W0Z2H1_9B URK xdhA UA14_02405, UA16 02328 Burkholderia multivorans 531 A0A1W1 A395 A0A1W1A395_9B URK xdhA UA19_02501, UA21 02488 Burkholderia multivorans 531 A0A1W0Z BE9 A0A1W0ZBE9_9B URK xdhA UA17_00017 Burkholderia multivorans 521 A0A1W1 A6N7 A0A1W1A6N7_9B URK xdhA UA18_02826 Burkholderia multivorans 531

[00136] D-glucuronate reductase (EC 1.1.1.19) is a known class of enzymes and, for example, a number of organisms, as outlined below, from the publicly accessible UniProt database:

Input Input name Gene names Body Length P14550 AK1A1_HUM AN AKR1A1 ALDR1, ALR Homo sapiens (Human) 325 Q9UGB7 MIOX_HUMA N MIOX ALDRL6, KSP32, RSOR Homo sapiens (Human) 285 Q9QXN5 MIOX_MOUS AND Miox Aldrl6, Rsor Mus musculus (mouse) 285 O35082 KLOT_MOUS AND Kl Mus musculus (mouse) 1,014 Q8WN98 MIOX PIG MIOX ALDRL6 Sus scrofa (pig) 282 Q9JII6 AK1A1_MOUS AND Akr1a1 Akr1a4 Mus musculus (mouse) 325 P51635 AK1A1_RAT Akr1a1 Alr Rattus norvegicus (Rat) 325 Q9QXN4 MIOX_RAT Miox Aldrl6, Ksp32, Rsor Rattus norvegicus (Rat) 285

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Input Input name Gene names Body Length P37769 KDUD_ECOLI kduD ygeC, yqeD, b2842, JW2810 Escherichia coli (K12 strain) 253 Q3ZCJ2 AK1A1_BOVI N AKR1A1 Bos taurus (Bovine) 325 Q3ZFI7 GAR1_HYPJE gar1 Hypocrea j ecorina (Trichoderma reesei) 309 Q5REY9 MIOX_PONAB MIOX Pongo abelii (Sumatran orangutan) (Pongo pygmaeus abelii) 285 P39160 UXUB_ECOLI uxuB b4323, JW4286 Escherichia coli (K12 strain) 486 H2PYX5 H2PYX5_PAN TR AKR1A1 Pan troglodytes (chimpanzee) 325 Q540D7 Q540D7_MOU SE Akr1a1 Akr1a4 Mus musculus (mouse) 325 H0ZCF8 H0ZCF8_TAE GU AKR1A1 Taeniopygia guttata (diamond-mandarin) (Poephila guttata) 327 K7FUR5 K7FUR5_PELS I AKR1A1 Pelodiscus sinensis (Chinese carapaceole turtle) (Trionyx sinensis) 327 F1PK43 F1PK43_CANL F AKR1A1 Canis lupus familiaris (Dog) (Canis familiaris) 325 M3VZ98 M3VZ98_FEL HERE AKR1A1 Felis catus (Cat) (Felis silvestris catus) 325 H0WVS3 H0WVS3_OTO GA AKR1A1 Otolemur garnettii (Small-eared Galago) (Dwarf rondo galago) 325 G1NT89 G1NT89_MYO LU AKR1A1 Myotis lucifugus (Small brown bat) 325 I3ML55 I3ML55_ICTT R AKR1A1 Ictidomys tridecemlineatus (Thirteen line squirrel) (Spermophilus tridecemlineatus) 325 H0VM25 H0VM25_CAV POWDER AKR1A1 Cavia porcellus (Guinea Pig) 325 M3YNR9 M3YNR9_MU SPF AKR1A1 Mustela putorius furo (Ferret) (Mustela furo) 325 G3W2S6 G3W2S6_SAR THERE IS AKR1A1 Sarcophilus harrisii (Tasmanian demon) (Sarcophilus laniarius) 325 U3K4S3 U3K4S3_FICA L AKR1A1 Ficedula albicollis (Collared Flycatcher) (Muscicapa albicollis) 327 A0A0D9S7F8 A0A0D9S7F8_ CHLSB AKR1A1 Chlorocebus sabaeus (Green monkey) (Cercopithecus sabaeus) 325 G1M4Y1 G1M4Y1_AIL ME AKR1A1 Ailuropoda melanoleuca (Pandagigante) 326 F6XYQ0 F6XYQ0_CAL ALREADY AKR1A1 Callithrix jacchus (White-tufted Marmoset) 325 W5NUN8 W5NUN8_SHE EP AKR1A1 Ovis aries (sheep) 325

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Input Input name Gene names Body Length A0A096N138 A0A096N138_ PAPAN AKR1A1 Papio anubis (Baboon) 325 H2N7K8 H2N7K8_PON AB AKR1A1 Pongo abelii (Sumatran orangutan) (Pongo pygmaeus abelii) 325 F6R8L7 F6R8L7_ORN AN AKR1A1 Ornithorhynchus anatinus (Platypus) 327 H9GLX5 H9GLX5_ANO HERE AKR1A1 Anolis carolinensis (Green anolis) (American chameleon) 358 U3II47 U3II47_ANAP L AKR1A1 Anas platyrhynchos (mallard) (Anas boschas) 328 F7CBN0 F7CBN0_HOR SE AKR1A1 Equus caballus (Horse) 324 I3L929 I3L929 PIG AKR1A1 Sus scrofa (pig) 326 F7GDV9 F7GDV9_MON OF AKR1A1 Monodelphis domestica (Short-tailed Cuíca) 325 G1NDE3 G1NDE3_MEL GA AKR1A1 Meleagris gallopavo (domestic turkey) 329 G3RAF6 G3RAF6_GOR GO AKR1A1 Gorilla gorilla gorilla (western lowland gorilla) 298 G3U0I8 G3U0I8_LOXA F AKR1A1 African loxodonta (savanna elephant) 325 V9HWI0 V9HWI0_HUM AN HEL-S-165mP HEL-S-6 Homo sapiens (Human) 325 A0A1V1TUJ3 A0A1V1TUJ3_ 9FUNG Water ANO14919 141650 Fungal species n ° 14919 838 B1AXW3 B1AXW3_MO USE Akr1a1 Mus musculus (mouse) 203 Q7CPT2 Q7CPT2_SALT Y STM3136 Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) 490

[00137] Uronolactonase is a known class of enzymes (EC 3.1.1.19) and catalyzes the reaction of D-glucurono-6,2-lactone + H (2) The <=> Dglucuronate and is also known as glucuronolactonase or D- glucurono6,2-lactone lactono hydrolase and a number of enzymes associated with this enzyme activity are known, see EC 3.1.1.19.

[00138] D-glucuronocinase (EC 2.7,1,43) is a class of enzymes known and, for example, Arabidopsis thaliana is involved in the biosynthesis of UDP-glucuronic acid (UDP-GlcA) with amino acids 126136 involved in the call. The polynucleotide used in this document can encode all or part of the amino acid sequence of SEQ ID NO: 29. [00139] Glucuronate-1-phosphate uridylyltransferase is a class of

Petition 870180132932, of 9/21/2018, p. 61/93 / 80 known enzymes (EC 2.7.7.44) that catalyzes the chemical reaction UTP + 1 phosphor-alpha-D-glucuronate to diphosphate + UDP-glucuronate and is known in the art, for example, from a number of organisms, as outlined below, from the publicly accessible UniProt database:

Input Input name Gene names Body Length Q9C5I1 USP_ARATH USP At5g52560, F6N7.4 Arabidopsis thaliana 614 Q5Z8Y4 USP_ORYSJ USP Os06g0701200, LOC_Os06g48760, OsJ_021664, P0596H10.4 Oryza sativa subsp. japonica (Rice) 616 A8HP64 A8HP64_CHLRE UAP2 CHLREDRAFT_32796 Chlamydomonas reinhardtii (Chlamydomonas smithii) 831 I1GWP2 I1GWP2_BRADI LOC100845164 Brachypodium distachyon (Brachypodium) (Trachynia distachya) 610 B9GTZ2 B9GTZ2_POPTR POPTR_0002s07790g Populus trichocarpa (Populus balsamifera subsp. Trichocarpa) 522 E0CR04 E0CR04 VITVI VIT 18s0001g01640 Vitis vinifera (Grape) 644 K4BT00 K4BT00_SOLLC Solanum lycopersicum (Tomato) (Lycopersicon esculentum) 617 A9TMZ5 A9TMZ5_PHYPA PHYPADRAFT_19655 1 Physcomitrella patens subsp. patens (Moss) 617 M1C415 M1C415_SOLTU Solanum tuberosum (Potato) 624 J3MHA9 J3MHA9 ORYBR Oryza brachyantha 611 W1NLN3 W1NLN3_AMBTC AMTR_s00202p00022 860 Amborella trichopoda 626 M5XAU8 M5XAU8_PRUPE PRUPE_ppa003010mg Prunus persica (Peach) (Amygdalus persica) 612 D7MS64 D7MS64_ARALL ARALYDRAFT_4953 27 Arabidopsis lyrata subsp. lyrata 614 A0A0J8D62 3 A0A0J8D623_BETVU BVRB_1g010300 Beta vulgaris subsp. vulgaris 620 B8B249 B8B249_ORYSI OsI_24356 Oryza sativa subsp. indica (Rice) 627 M8BEG3 M8BEG3_AEGTA F775_26791 Aegilops tauschii (Aegilops squarrosa) 583 I1Q4Z2 I1Q4Z2_ORYGL Oryza glaberrima (African rice) 616 M7ZGA3 M7ZGA3_TRIUA TRIUR3_12743 Triticum urartu (Crithodium urartu) 592 A0A0D3DV G6 A0A0D3DVG6_BRAO L Brassica oleracea var. oleracea 619 M4CUA3 M4CUA3_BRARP Brassica rapa subsp. pekinensis (Couvechina) (Brassica pekinensis) 618

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Input Input name Gene names Body Length A0A0E0Q28 5 A0A0E0Q285_ORYR U Oryza rufipogon (Brown rice) 616 A0A0E0HU Y3 A0A0E0HUY3_ORYN I Oryza nivara 616 A0A061FH A6 A0A061FHA6_THEC Ç TCM_035220 Theobroma cacao (cocoa) 621 W5I0D4 W5I0D4_WHEAT Triticum aestivum (Wheat) 625 A0A0E0AD Y6 A0A0E0ADY6_9ORY Z Oryza glumipatula 616 A0A0D3GK G7 A0A0D3GKG7_9ORY Z Oryza barthii 616 A0A0D9WT S5 A0A0D9WTS5_9ORY Z Leersia perrieri 624

[00140] UDP-D-glucose dehydrogenase (EC 1.1.1.22) is a known class of enzymes that catalyzes the chemical reaction UDP-glucose + 2 NAD + + H2O-glucuronate of UDP + 2 NADH + 2 H + (EC 1.1.1.22 ) and may include all or part of SEQ ID NO: 30.

[00141] Other strings from the publicly available database of UniProt are:

O33952, Q04872, Q7DBF9, UDG_ECO57; UDG8_ECOLX; UDG_ECO11; Q8FG45, P76373, O86422, UDG_PSEAE; UDG_ECOL6; UDG_ECOLI; O54068, Q1RKF8, Q92GB1, UDG_RICCN; UDG_RHIME; UDG_RICBR; Q4UK39, O05973, Q68VX0, UDG_RICTY; UDG_RICFE; UDG_RICPR; Q04873, P37791, Q57346, UDG_STREE; UDG_SALTY; UDG_SHIFL; P0C0F5, P0DG68, Q5X9A8, UDG_STRP6; UDG_STRP1; UDG_STRP3; Q8NKX0, P0DG69, P0C0F4, UDG_STRPY; UDG_STRP8; UDG_STRPQ; Q9FZE1, Q75GS4, Q96558, UGDH1_ARATH; UGDH1_ORYSJ; UGDH1_SOYBN;

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Q9LIA8, B7F958, Q9LF33, UGDH2_ARATH; UGDH2_ORYSJ; UGDH3_ARATH; Q9AUV6, Q9FM01 Q2QS14, UGDH3_ORYSJ; UGDH4_ARATH; UGDH4_ORYSJ; Q2QS13, P12378, Q19905, UGDH5_ORYSJ; UGDH_BOVIN; UGDH_CAEEL; Q5F3T9, O02373, O60701, UGDH_CHICK; UGDH_DROME; UGDH_HUMAN; O70475, UGDH_MOUSE; O34862, YTCA_BACSU; Q5R7B3, UGDH_PONAB; P96718, YWQF_BACSU; O70199, UGDH_RAT;

[00142] UTP-glucose-1-phosphate uridylyltransferase (EC 2.7.7.9) is a known class of enzymes, also known as glucose-1-phosphate uridylyltransferase (or UDP-glucose pyrophosphorylase), is an enzyme involved in carbohydrate metabolism . This synthesizes UDP Glucose from glucose-1-phosphate and UTP. There are hundreds of known sequences of various species associated with this class in the art.

[00143] Phosphoglucomutase (CE 5.4.2.2) is a known class of enzymes and is an enzyme that transfers a phosphate group on an α-D-glucose monomer from position 1 'to position 6' in the forward direction or in position 6 'to position 1' in the reverse direction. There are hundreds of known sequences of various species associated with this class in the art. [00144] Hexokinase (EC 2.7.1.1) is a class of known enzymes that phosphorylates hexoses (six-carbon sugars), forming hexose phosphate. There are hundreds of known sequences of various species associated with this class in the art.

[00145] Assay of hydroxylation degree of proline residues in recombinant protein (for example, collagen). The degree of hydroxylation of

Petition 870180132932, of 9/21/2018, p. 64/93 / 80 proline residues in recombinant protein (e.g., collagen), produced according to the present invention, can be assayed by known methods, including by mass spectrometry with liquid chromatography, as described by Chan, et al. Biotechnology BMC 12:51 (2012), which is incorporated by reference.

[00146] Assay of the degree of hydroxylation of lysine residues in the recombinant protein (for example, collagen). Lysine hydroxylation and collagen crosslinking is described by Yamauchi, et al., Methods in Molecular Biology, vol. 446, pages 95-108 .; Humana Press (2008), which is incorporated by reference. The degree of hydroxylation of lysine residues into recombinant protein (eg, collagen), produced according to the present invention, can be tested by known methods, including the method described by Hausmann, Biochimica et Biophysica Acta (BBA) - Protein Structure 133 (3): 591-593 (1967) which is incorporated by reference.

[00147] Degree of hydroxylation. The degree of hydroxylation of lysine or proline residues in the protein (for example, collagen), produced according to the present invention, is preferably at least 10%, 15%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100%. Also within the scope of the present invention, the degree of hydroxylation of lysine residues plus proline is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55 %, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100%.

[00148] Melting point of collagen. The degree of hydroxylation of the lysine, proline or lysine and proline residues in the collagen can be estimated by the melting temperature of a hydrated collagen, such as a hydrogel, as compared to a control collagen having a known content of hydroxylated amino acid residues. The melting temperatures of collagen can vary between 25 and 40oC with more highly hydroxylated collagens having, in general, higher melting temperatures. This range includes

Petition 870180132932, of 9/21/2018, p. 65/93 / 80 all subintervals and intermediate values, including subintervals that are connected at the lower and upper end by a temperature selected from 25, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 , 38, 39 and 40 ° C.

[00149] Codon modification. Within the scope of the present invention, it is envisaged that the genetic sequence introduced into the yeast host cell will be modified from its native sequence. This process includes changing a polynucleotide sequence that encodes the protein of interest (for example, collagen, such as the collagen DNA sequence found in nature), to modify the amount of recombinant protein (for example, collagen) expressed by a yeast, such as Pichia pastoris, to modify the amount of recombinant protein (eg, collagen) secreted by the recombinant yeast, to modify the rate of expression of recombinant protein (eg, collagen) in the recombinant yeast or to modify the degree of hydroxylation of proline or lysine residues in the recombinant protein (eg, collagen). Codon modification can also be applied to other proteins, such as hydroxylases, for similar purposes or to target hydroxylases to particular intracellular or extracellular compartments, for example, to target a proline hydroxylase to the same compartment, such as the endoplasmic reticulum, as a molecule of recombinant protein (eg, collagen). Codon selections can be made based on the effect on the secondary structure of RNA, effect on transcription and gene expression, effect on the speed of translation elongation and / or the effect on protein envelope.

[00150] Codons encoding collagen or hydroxylase can be modified to reduce or increase the secondary structure in mRNA encoding recombinant collagen or hydroxylase or can be modified to replace a redundant codon with a codon that, in

Petition 870180132932, of 9/21/2018, p. 66/93 / 80 medium, is used most frequently by a yeast host cell in all yeast protein coding sequences (eg codon sampling), is used less frequently by a yeast host cell based on all protein coding sequences in yeast (for example, codon sampling), or redundant codons that appear in proteins that are abundantly expressed by yeast host cells or that appear in proteins that are secreted by yeast host cells (for example , a codon selection based on a High Codon Adaptation Index that makes the gene "look like" a highly expressed gene or gene that encodes a secretible expression host protein).

[00151] The codon modification can be applied to all or part of a protein coding sequence, for example, applied to at least one of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth 10% of a coding sequence or combinations thereof. It can also be applied selectively to a codon encoding a particular amino acid or to codons encoding some but not all of the amino acids that are encoded by redundant codons. For example, only codons for leucine and phenylalanine can be modified per codon, as described above. Amino acids encoded by more than one codon are described by the codon table, which is known in the art.

[00152] Codon modification includes the so-called codon optimization methods described at https://www.atum.bio/services/genegps (last accessed on July 13, 2017), by https://www.idtdna.com / CodonOpt or Villalobos, Alan et al. BMC Bioinformatics 7 (2006): 285. PMC. August 17, 2017, incorporated herein by reference.

[00153] Codon modification also includes selection of codons to allow the formation of secondary mRNA structure or the

Petition 870180132932, of 9/21/2018, p. 67/93 / 80 minimize or eliminate the secondary structure. An example of this is to make codon selections in order to eliminate, reduce or weaken the strong secondary structure of the secondary structure at or around a ribosome binding site or initiation codon.

[00154] In one embodiment of the present invention, the sequence of the α-ketoglutarate transporter gene is optimized for use in yeasts, particularly Pichia. An example of an optimized α-ketoglutarate transporter (kgtP) gene is defined in SEQ ID NO: 3.

[00155] Collagen Fragments. A recombinant collagen molecule can comprise a fragment of the amino acid sequence of a native collagen molecule capable of forming tropocolagen (trimeric collagen) or a modified collagen molecule or collagen molecule truncated with an amino acid sequence of at least 70, 80, 90 , 95, 96, 97, 98 or 99% identical or similar to a native collagen amino acid sequence (or to a fibril-forming region thereof or to a segment substantially comprising [Gly-XY] n), such as those of amino acid sequences of Col1A1 Col1A2 and Col3A1, described by accession numbers NP_001029211.1 (SEQ ID NO: 11) (https://www.ncbi.nlm.nih.gov/protein/77404252, last accessed on 23 August 2017), NP_776945.1 (SEQ ID NO: 12) (https://www.ncbi.nlm.nih.gov/protein/27806257 last accessed on 23 August 2017) and NP_001070299.1 (SEQ I NO: 13) (https://www.ncbi.nlm.nih.gov/protein/116003881 last accessed on 9 February 2017), respective which are incorporated by reference. [00156] A gene encoding collagen or a hydroxylase can be truncated or otherwise modified to add or remove sequences. Such modifications can be made to customize the size of a polynucleotide or vector, to target the expressed protein to the endoplasmic reticulum or other cellular or extracellular compartment, or

Petition 870180132932, of 9/21/2018, p. 68/93 / 80 to control the length of an encoded protein. For example, the inventors have found that constructions containing only the Pre sequence generally work better than those containing the entire Prepro sequence. The Pre sequence was fused to P4HB to locate P4HB in the ER, where collagen is also located.

[00157] Modified coding sequences for collagens and hydroxylases. A polynucleotide coding sequence for collagen or hydroxylase, or other proteins, can be modified to encode a protein that is at least 70, 80, 90, 95, 96, 97, 98 or 100% identical or similar to a known amino acid sequence and which retains the essential properties of the unmodified molecule, for example, the ability to form tropocholagen or the ability to hydroxylase lysine or proline residues in collagen. Glycosylation sites on a collagen molecule can be removed or added. Modifications can be made to facilitate collagen yield or its secretion by a yeast host cell or to change its structural, functional or aesthetic properties. A modified hydroxylase or collagen coding sequence can also be codon-modified, as described in this document.

[00158] BLASTN can be used to identify a polynucleotide sequence with at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98 %, 99% or <100% sequence identity to a reference polynucleotide, such as a polynucleotide that encodes a collagen, one or more hydroxylases described herein, or signal, leader or secretion peptides or any other proteins disclosed herein . A representative BLASTN configuration modified to find highly similar strings used an Expect Threshold of 10 and a Wordsize of 28, maximum matches in the query interval of 0, match / mismatch scores of 1 / -2 and cost of

Petition 870180132932, of 9/21/2018, p. 69/93 / 80 linear gap. Low complexity regions can be filtered or masked. The standard configurations of a standard nucleotide BLAST are known in the art.

[00159] BLASTP can be used to identify an amino acid sequence with at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% or <100% sequence identity or similarity to a reference amino acid, such as a collagen amino acid sequence, using a similarity matrix, such as BLOSUM45, BLOSUM62 or BLOSUM80, in which BLOSUM45 can be used for approximately related sequences, BLOSUM62 for medium interval sequence and BLOSUM80 for more distantly related sequences. Except where indicated, a similarity score will be based on the use of BLOSUM62. When using BLASTP, the percentage similarity is based on the positive BLASTP score and the percentage string identity is based on the BLASTP identity score. BLASTP identities show the number and fraction of total residues in pairs of high-score strings that are identical; and BLASTP Positives shows the number and fraction of residues for which the alignment scores have positive values and are similar to each other. Amino acid sequences with these degrees of identity or similarity or any intermediate degree of identity or similarity with the amino acid sequences disclosed in this document are contemplated and covered by this disclosure. A representative BLASTP configuration that uses an Expect Threshold of 10, a Word Size of 3, BLOSUM 62 as a matrix and Gap Penalty of 11 (Existence) and 1 (Extension) and a conditional composition score matrix adjustment. Other standard configurations for BLASTP are known in the art.

[00160] The term variant, modified sequence or analog, as applied to the polypeptides disclosed in this document, refers to

Petition 870180132932, of 9/21/2018, p. 70/93 / 80 a polypeptide comprising an amino acid sequence that is at least 70, 80, 90, 95 or 99% identical or similar to the amino acid sequence of a biologically active molecule. In some embodiments, the derivative comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of a native or previously manipulated sequence. The derivative may comprise additions, deletions, substitutions or a combination thereof to the amino acid sequence of a native or previously engineered molecule. For example, a derivative can incorporate or eliminate 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more lysine or proline residues compared to a native collagen sequence. Such selections can be made to modify the clearance or tightness of a recombinant tropocholagen or fibrillated collagen.

[00161] A derivative may include a mutant polypeptide having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-15, 16-20, 21-25 or 26-30 additions, substitutions or eliminations of amino acid residues. Additions or substitutions also include the use of non-naturally occurring amino acids or modified amino acids. A derivative can also include chemical modifications to a polypeptide, such as crosslinking between cysteine residues, or hydroxylated or glycosylated residues.

[00162] Yeast strains. The present invention uses yeast to produce collagen or other proteins or carbohydrates. In particular, the present invention uses modified yeasts to produce collagen or other carbohydrates with an increased degree of hydroxylation. Yeast can be manipulated to produce or overproduce α-ketoglutarate and / or ascorbic acid by inserting one or more of a gene expression for a kgtP gene, a gene that expresses a hydroxylase or gene (s) needed to complete a functional ascorbate synthesis pathway.

Petition 870180132932, of 9/21/2018, p. 71/93 / 80 [00163] Suitable yeast includes, but is not limited to, those of the genus Pichia, Candida, Komatagaella, Hansenula, Cryptococcus, Saccharomyces and combinations of these. Yeast can be modified or hybridized. Hybridized yeast is a mixed breed of different strains of the same species, different species of the same genus or strains of different genera. Examples of yeast strains that can be used according to the invention include Pichia pastoris, Pichia membranifaciens, Pichia deserticola, Pichia cephalocereana, Pichia eremophila, Pichia myanmarensis, Pichia anomala, Pichia nakasei, Pichia siamensi, Pichia barisi Pichia norvegensis, Pichia thermomethanolica, Pichia stipites, Pichia subpelliculosa, Pichia exigua, Pichia occidentalis, Pichia cactophila, Saccharomyces cerevisiae, Saccharomyces pombe, and the like. [00164] Pichia pastoris is a species of yeast that was used to express biotherapeutic proteins recombinantly, such as human gamma interferon, see Razaghi, et al., Biologicals 45: 52-60 (2017) It was used to express collagen type III and prolyl-4-hydroxylase, see Vuorela, et al., EMBO J. 16: 6702-6712 (1997). Collagen and prolyl-4 hydroxylase have also been expressed in Escherichia coli to produce collagenous material, see Pinkas, et al., ACS Chem. Biol. 6 (4): 320-324 (2011).

[00165] In one embodiment, the invention is directed to strains of Pichia pastoris that have been manipulated to express collagen, hydroxylase (s), α-ketoglutarate transporter and / or gene (s) needed to complete a functional ascorbate synthesis pathway . Useful host strains of Pichia pastoris include, but are not limited to, wild type (PPS-9010 strain); aox1A (MutS) (strain PPS-9011) which is a methanol-derived derivative of PPS-9010; and pep4A, prb1A (strain PPS-9016) which is deficient in protease. These strains are publicly available and can be obtained from TUNA at https://www._atum.bio/products/cell-strains.

Petition 870180132932, of 9/21/2018, p. 72/93 / 80 [00166] Polypeptide secretion sequences for yeast. In some embodiments, a polypeptide that is encoded by a yeast host cell is fused to a polypeptide sequence that facilitates its secretion from the yeast. For example, a vector can encode a chimeric gene comprising a coding sequence for the fused collagen with a sequence encoding a secretion peptide. Secretion sequences that can be used for this purpose include Prepro sequence of Saccharomyces alpha mating factor, Pre sequence of Saccharomyces alpha mating factor, PHO1 secretion signal, Aspergillus niger α-amylase signal sequence, Glucoamylase signal sequence from Aspergillus awamori, Homo sapiens serum albumin signal sequence, Kluyveromcyes maxianus inulinase signal sequence, Saccharomyces cerevisiae invertase signal sequence, Saccharomyces cerevisiae killer protein signal sequence and Gallus gallus gallus lysozyme signal sequence. Other secretion sequences known in the art can also be used.

[00167] Yeast terminators and promoters. In some embodiments, one or more of the following yeast promoters can be incorporated into a vector to promote the transcription of mRNA that encodes the protein of interest (eg, collagen), hydroxylase (s), α-ketoglutarate transporter and / or gene (s) needed to complete a functional ascorbate synthesis pathway. Promoters are known in the art and include pAOX1, pDas1, pDas2, pPMP20, pCAT, pDF, pGAP, pFDH1, pFLD1, pTAL1, pFBA2, pAOX2, pRKI1, pRPE2, pPEX5, pDAK1, pFGH1, pFPHP, p1 pPFK1, pGPM1 and pGCW14.

[00168] In some embodiments, a yeast terminator sequence is incorporated into a vector to terminate the mRNA transcription encoding the protein of interest (eg, collagen), hydroxylase (s), α-ketoglutarate transporter and / or gene (s) needed to complete

Petition 870180132932, of 9/21/2018, p. 73/93 / 80 a functional ascorbate synthesis pathway. Terminators include, but are not limited to, AOX1 TT, Das1 TT, Das2 TT, AOD TT, PMP TT, Cat1 TT, TPI TT, FDH1 TT, TEF1 TT, FLD1 TT, GCW14 TT, FBA2 TT, ADH2 TT, FBP1 TT and GAP TT.

[00169] Peptidases in addition to pepsin. Pepsin can be used to process collagen into tropolagen by removing sequences from the N-termini and C-termini. Other proteases, including but not limited to, collagenase, trypsin, chymotrypsin, papain, ficin and bromelain, can also be used for this purpose . As used in this document, “stable collagen” means that, after being exposed to a particular concentration of pepsin or other protease, at least 20, 30, 40, 50, 60, 75, 80, 85, 90, 95 or 100% of initial collagen concentration is still present. Preferably, at least 75% of a stable collagen will remain after treatment with pepsin or another protease as compared to an unstable collagen treated under the same conditions for the same period of time. Prior to post-translational modification, collagen is not hydroxylated and degrades in the presence of a high concentration of pepsin (for example, a ratio of pepsin: protein of 1: 200 or more).

[00170] Once post-translationally modified, a collagen can be placed in contact with pepsin or another protease to cleave the collagen's N-terminal and C-terminal propeptides, thus allowing collagen fibrillation. Hydroxylated collagen has better thermostability compared to non-hydroxylated collagen and is resistant to digestion of high concentration of pepsin, for example, in a ratio of pepsin: total protein from 1:25 to 1: 1. Therefore, to avoid premature proteolysis of recombinant collagen, it is useful to provide hydroxylated collagen.

[00171] Alternative expression systems. Collagen and other proteins can be expressed in yeast cell types other than Pichia pastoris, for example, they can be expressed in another yeast,

Petition 870180132932, of 9/21/2018, p. 74/93 / 80 methylotrophic yeast or other organism. Saccharomyces cerevisiae can be used with any of a large number of expression vectors. Expression vectors commonly used are transfer vectors containing the 2P origin of replication for propagation in yeast and the Col E1 origin for E. coli, for efficient transcription of the foreign gene. A typical example of such vectors based on 2P plasmids is pWYG4, which has the 2P ORI-STB elements, the GAL1-10 promoter and the 2P D gene terminator. In this vector, an Ncol cloning site is used to insert the gene for the polypeptide to be expressed and to provide an ATG initiation codon. Another expression vector is pWYG7L, which has 2aORI, STB, REP1 and REP2 intact and the GAL1-10 promoter and uses the FLP terminator. In this vector, the coding polynucleotide is inserted into the polylinker with its 5 'ends at a BamHI or Ncol site. The vector containing the inserted polynucleotide is transformed into S. cerevisiae or after removal of the cell wall to produce spheroplasts that absorb DNA in the treatment with calcium and polyethylene glycol or by treating intact cells with lithium ions.

[00172] Alternatively, DNA can be introduced by electroporation. Transformants can be selected, for example, using host yeast cells that are auxotrophic for leucine, tryptophan, uracil or histidine in conjunction with selectable marker genes, such as LEU2, TRP1, URA3, HIS3 or LEU2-D.

[00173] There are several genes responsive to methanol in methylotrophic yeasts, such as Pichia pastoris, and the expression of each is controlled by regulatory regions responsive to methanol, also referred to as promoters. Any such promoter responsive to methanol is suitable for use in the practice of the present invention. Examples of specific regulatory regions include the AOX1 promoter, the AOX2 promoter, dihydroxyacetone synthetase (DAS), the P40 promoter and the promoter

Petition 870180132932, of 9/21/2018, p. 75/93 / 80 for the P. pastoris catalase gene etc.

[00174] Methylotrophic yeast Hansenula polymorpha is also mentioned. Growth in methanol results in the induction of key enzymes of methanol metabolism, such as MOX (methanol oxidase), DAS (dihydroxyacetone synthase) and FMHD (dehydrogenase format). These enzymes can make up to 30 to 40% of the total cellular protein. The genes encoding the production of MOX, DAS and FMDH are controlled by strong promoters induced by growth in methanol and repressed by growth in glucose. Any or all of these three promoters can be used to obtain high-level expression of heterologous genes in H. polymorpha. Therefore, in one aspect, a polynucleotide that encodes animal collagen, fragments or variants thereof, is cloned into an expression vector under the control of an inducible H. polymorpha promoter gene. If product secretion is desired, a polynucleotide that encodes a signal sequence for yeast secretion is fused in-frame with the polynucleotide. In an additional embodiment, the expression vector preferably contains an auxotrophic marker gene, such as URA3 or LEU2, which can be used to supplement an auxotrophic host deficiency.

[00175] The expression vector is then used to transform H. polymorpha host cells using techniques known to those skilled in the art. A useful transformation resource for H. polymorpha is the spontaneous integration of up to 100 copies of the expression vector within the genome. In most cases, the integrated polynucleotide forms multimers exhibiting a head-tail arrangement. The integrated foreign polynucleotide has been shown to be mitotically stable in several recombinant strains, even under non-selective conditions. This phenomenon of high copy integration adds further to the high productivity potential of the system. [00176] Foreign DNA is inserted into the yeast genome or kept

Petition 870180132932, of 9/21/2018, p. 76/93 / 80 episomally to produce collagen. The DNA sequence for collagen is introduced into the yeast via a vector. Foreign DNA is any non-yeast host DNA and includes, for example, but is not limited to, mammals, Caenorhabditis elegans and bacteria. Mammalian DNA suitable for yeast collagen production includes, but is not limited to, bovine, porcine, kangaroo, crocodile, elephant, giraffe, zebra, llama, alpaca, lamb, dinosaur and combinations of these.

[00177] The DNA to allow the production of ascorbate can also be inserted into a single or multiple vectors. For a plant pathway, the DNA of the genes GDP-L-Gal phosphorylase, Inositol phosphate phosphate, GDPManosis-3,5-epimerase are inserted through a vector into the yeast cell. Pichia is already known to contain the remaining genes in the pathway for ascorbate production from glucose transformed through a vector into the yeast cell.

[00178] The DNA for the ascorbic pathway can be inserted alone or combined with the DNA for proteins. The ascorbic pathway allows the production of healthy yeast strains that are suitable for producing most proteins.

[00179] The DNA to allow the production of the α-ketoglutarate transporter can also be inserted into a single vector. The yeast-optimized kgtP gene is transformed through a vector in the yeast cell.

[00180] The DNA for the α-ketoglutarate transporter can be inserted alone or combined with DNA for proteins or carbohydrates. The α-ketoglutarate transporter allows for healthy, fast-growing yeast strains that are suitable for producing most proteins and carbohydrates. The carrier also allows for increased production of hydroxylated collagen.

Petition 870180132932, of 9/21/2018, p. 77/93 / 80 [00181] DNA can be inserted into a vector. Vectors useful for the expression of proteins in yeasts are known, see Ausubel et al., Supra, Vol. 2, Chapter 13; Grant et al. (1987) Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Ed. Wu & Grossman, Acad. Press, NY 153: 516-544; Glover (1986) DNA Cloning, vol. II, IRL Press, Wash., D.C., Ch. 3; Bitter (1987) Heterologous Gene Expression in Yeast, in Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y. 152: 673684; and The Molecular Biology of the Yeast Saccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II (1982), the disclosures of which are incorporated into this document by reference. Other suitable vectors include, but are not limited to, pHTX1- BiDi-P4HA-Pre-P4HB hygro, pHTX1- BiDi-P4HA-PHO1-P4HB hygro, pGCW14-pGAP1- BiDi-P4HAPrepro-P4HB G418, pGCWD-pG1- P4HA-PHO1-P4HB Hygro, Zeoci modified by pDF-Col3A1, Zeocin modified by pCAT-Col3A1, Zeoci modified by pDF-Col3A1 with AOX1 landing, pHTX1- BiDiP4HA-Pre-Pro-P4HB hygro. The vectors normally included at least one restriction site for DNA linearization.

[00182] A selected promoter can improve the production of a recombinant protein and can be included in a vector comprising sequences that encode the protein of interest (for example, collagen) or hydroxylases. Promoters suitable for use in the present invention include, but are not limited to, AOX1 methanol-induced promoter, pDF-repressed promoter, pCAT-repressed promoter, Das1-Das2 methanol-induced bidirectional promoter, bidT-directional constitutive promoter pHTX1, pGCW bidirectional constitutional promoter pGCW14 pGAP1 and combinations of these. Suitable methanol-induced promoters include, but are not limited to AOX2, Das 1, Das 2, pDF, pCAT, pPMP20, pFDH1, pFLD1, pTAL2, pFBA2, pPEX5, pDAK1, pFGH1, pRKI1, pREP2 and one of these.

Petition 870180132932, of 9/21/2018, p. 78/93 / 80 [00183] As mentioned above, it is also envisaged that a vector can be manipulated to be a multifunctional vector that contains each of the selected genes from at least one, including any combination or sub-combination of the same or all of them, collagen, hydroxylase (s), α-ketoglutarate transporter and / or gene (s) needed to complete a functional ascorbate synthesis pathway. [00184] In the vectors according to the invention, including the multifunctional vector, a terminator can be placed at the end of each open reading frame used in the vectors incorporated in the yeast. The DNA sequence for the terminator is inserted into the vector. For replication vectors, a source of replication is required to initiate replication. The DNA sequence for the origin of replication is inserted into the vector. One or more DNA sequences containing homology to the yeast genome can be incorporated into the vector to facilitate recombination and incorporation into the yeast genome or to stabilize the vector once transformed into the yeast cell.

[00185] A vector according to the invention will also generally include at least one selective marker that is used to select yeast cells that have been successfully transformed. Markers are sometimes related to antibiotic resistance and markers can also be related to the ability to grow with or without certain amino acids (auxotrophic markers). Suitable auxotrophic markers included, but are not limited to, ADE, HIS, URA, LEU, LYS, TRP and combinations of these. To provide selection of yeast cells containing a recombinant vector, at least one DNA sequence for a selection marker is incorporated into the vector.

[00186] In some embodiments of the invention, amino acid residues, such as lysine and proline, in a protein expressed by recombinant yeast, including collagen or protein similar to

Petition 870180132932, of 9/21/2018, p. 79/93 / 80 collagen, may lack hydroxylation or may have a degree of hydroxylation greater or less than a natural or unmodified protein, for example, collagen or collagen-like protein. In other embodiments, amino acid residues in a protein expressed by recombinant yeast, including collagen or collagen-like protein, may lack glycosylation or have a degree of glycosylation less or greater than a natural or unmodified collagen or protein similar to example, collagen or collagen-like protein.

[00187] Hydroxylated collagen, for example, has a higher melting temperature (> 37 ^o C) than under hydroxylated collagen (<32 ^o C) and also fibrils better than under hydroxylated collagen and forms a stronger structure for purposes materials. The melting temperature of a collagen preparation can be used to estimate its degree of hydroxylation and can vary, for example, from 25 to 40 ^o C, as well as all intermediate values, such as 25, 26, 27, 28, 29 , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40 ^o C, as well as the subintervals that are connected at the lower and upper end by a temperature selected from the previous values can be used to select a collagen population for use.

[00188] Under hydroxylated collagen, only a material similar to gelatin may be formed, not suitable for durable items, such as shoes or bags, but which can be formulated in softer or more absorbent products. By increasing the degree of hydroxylation, it is possible to improve the thermal stability of collagen and it can also improve the strength of the biofabricated material produced from it. These biofabricated materials may be more suitable for items that require greater durability, including shoes, bags, seats, etc. As a means to modulate the degree of hydroxylation, it is envisaged that the number of copies or the expression of the respective enzymes can be increased, it is also envisaged to modify the temperature, pH and carbon source of the cell culture.

Petition 870180132932, of 9/21/2018, p. 80/93 / 80 [00189] The engineered yeast cells described above can be used as hosts to produce collagen or other proteins or carbohydrates. For this, the cells are placed in media inside a fermentation chamber and fed with dissolved oxygen and a carbon source, under controlled pH conditions, for a period of time ranging from twelve hours to 1 week. Suitable media include, but are not limited to, complex buffered medium containing glyceral (BMGY), complex buffered medium containing methanol (BMMY) and yeast peptone dextrose extract (YPD). Due to the fact that protein is produced in the yeast cell and using collagen as an example, in order to isolate the collagen, one must use a secretory yeast strain or lyse the yeast cells to release the collagen. The collagen can then be purified using conventional techniques, such as centrifugation, precipitation, filtration, chromatography and the like.

EXAMPLES [00190] The following non-limiting examples are illustrative of the present invention. The scope of the invention is not limited to the details described in these Examples.

EXAMPLE 1 [00191] Yeast modification to allow a functional plant synthesis pathway for ascorbate-strain of Pichia pastoris BG10 (wild type) was obtained from TUMA (formerly DNA 2.0). A vector containing DNA sequences of GDP-L-Gal phosphorylase (SEQ ID NO: 19), Inositol phosphate phosphate (SEQ ID NO: 20) and GDP-Mannose-3,5-epimerase (SEQ ID NO: 18) was inserted in wild type Pichia pastoris to generate a modified strain. The DNA was digested by Pme I and transformed into PP1 (wild type Pichia pastoris strain). This was suitable as a host strain. EXAMPLE 2 [00192] The host strain of example 1 is modified as

Petition 870180132932, of 9/21/2018, p. 81/93 / 80 described below:

[00193] The DNA encoding native Type III bovine collagen is sequenced and the sequence is amplified by the polymerase chain reaction protocol "PCR" to create a linear DNA sequence.

[00194] The DNA encoding the P4HA / B enzymes is sequenced (SEQ ID NO: 8 and SEQ ID NO: 9) and the sequence is amplified by the PCR polymerase chain reaction protocol to create a linear DNA sequence.

[00195] DNA is transformed into the strain of example 5 using an Electroporation Protocol in Pichia (Total System # 1652660 from Bio-Rad Gene Pulser XcellTM). Yeast cells are transformed with coexpression plasmid P4HA / B and transformants selected on Hygro plate (200 µg / ml).

[00196] A single colony of the strain resulting from example 1 is inoculated in 100 ml of YPD medium and grown at 30 degrees overnight with agitation at 215 rpm. The next day, when the culture reached OD600 ~ 3.5 (~ 3-5 X 10 ⁷ cells / OD600), it is diluted with fresh YPD to OD600 ~ 1.7 and grown for another hour at 30 degrees with agitation at 215 rpm.

[00197] The cells are centrifuged at 3,500g for 5 min; washed once with water and resuspended in 10 ml of 10 mM Tris-HCl (pH 7.5), 100 mM LiAc, 10 mM DTT (added fresh) and 0.6 M Sorbitol.

[00198] For each transformation, 8 X 10 ⁸ cells are aliquoted in 8 ml of 10 mM Tris-HCl (pH 7.5), 100 mM LiAc, 10 mM DTT, 0.6 M Sorbitol and allowed to incubate at room temperature for 30 min.

[00199] The cells are centrifuged at 5000g for 5 min and washed with 1.5 ml of ice-cold 1M Sorbitol 3 times and resuspended in 80 ul of ice-cold 1M Sorbitol.

[00200] Various amounts (about 5 pg) of linearized DNA are added to the cells and mixed by pipetting.

Petition 870180132932, of 9/21/2018, p. 82/93 / 80 [00201] The cells and the DNA mixture (80-100 pL) are added to a 0.2 cm electroporation cuvette and pulsed using Pichia - protocol (1500 v, 25 uF, 200Ω).

[00202] The cells are immediately transferred to 1 ml of YPD and 1M Sorbitol mixture (1: 1) and incubated at 30 degrees for> 2 hours. [00203] These cells are plated at different densities and incubated at 30 ° C for 2 days.

[00204] The single colonies that appeared on the plate after two days were inoculated in 2 mL of BMGY medium in a 24-well deep plate and cultured for at least 48 hours at 30 degrees Celsius with agitation at 900 rpm. The resulting cells were tested for collagen using cell lysis, SDS-page and pepsin assay following the procedure below.

[00205] Yeast cells are lysed in 1x lysis buffer using a Qiagen TissueLyser at a speed of 30 Hz continuously for 14 min. The lysis buffer is made up of 2.5 ml of 1 M HEPES (50 mM final concentration); 438.3 mg NaCl; final concentration 150 mM; 5 ml of glycerol; final concentration 10%; 0.5 ml of Triton X-100; final concentration 1%; and 42 ml of Millipure water.

[00206] The lysed cells are centrifuged at 2,500 rpm at 4 ° C for 15 minutes in a table centrifuge. The supernatant is retained and the pellet discarded.

[00207] The SDS-PAGE in the presence of 2-mercaptoethanol is performed on the supernatant, molecular weight markers, negative control and a positive control. After electrophoresis, the gel is removed and stained with Commassie Blue and then discolored in water.

[00208] A pepsin test is performed with the following procedure:

Before treatment with pepsin, the BCA assay is performed

Petition 870180132932, of 9/21/2018, p. 83/93 / 80 to obtain the total protein of each sample according to the Thermo Scientific protocol. Total protein is normalized to the lowest concentration for all samples. (Note: if the lowest total protein concentration is less than 0.5mg / mL, do not use this concentration for normalization).

[00209] 100 µL of lysate is placed in a microcentrifuge tube. A main mixture is created containing the following: 37% HCl (0.6pL of acid per 100 pL) and Pepsin (stock is 1mg / mL in deionized water and the final addition of pepsin should be in the proportion of 1:25 pepsin: total protein (weight: weight) After adding pepsin, mix 3X with a pipette and allow the samples to incubate for one hour at room temperature for the pepsin reaction to occur. ^ After one hour, add volume 1: 1 of LDS loading buffer containing β-mercaptoethanol for each sample and incubation for 7 minutes at 70oC is allowed. (In this situation, 100uL of LDS should be added.) Then, it is rotated at 14,000 rpm for 1 minute to remove turbidity.

[00210] Add 18 µL of the top of the sample in a 3-8% TAE buffer and run the gel for 1h 10 minutes at 150V.

EXAMPLE 3

Yeast producing ascorbate [00211] The cells of Example 1 are inoculated into medium containing glucose and cultured at 30 degrees C with agitation. The samples are collected at different times (for example, 24, 48, 72 hours, etc.) and analyzed for ascorbic acid, both intracellular and extracellular, using a commercially available kit. The amount of ascorbic acid is constant over time.

EXAMPLE 4

Yeast producing ascorbate and hydroxylated collagen [00212] The cells of Example 2 are inoculated in medium containing

Petition 870180132932, of 9/21/2018, p. 84/93 / 80 glucose and cultured at 30 degrees C with agitation. The samples are collected at different times (for example, 24, 48, 72 hours, etc.) and analyzed for ascorbic acid, both intracellular and extracellular, using a commercially available kit. The level of collagen expression and hydroxylation are also analyzed by known methods.

EXAMPLE 5 [00213] Yeast modification to include an α-ketoglutarate transporter. The strain Pichia pastoris (wild type) was obtained from TUNA (formerly DNA 2.0).

[00214] A vector containing kgtP DNA sequences (SEQ ID NO: 3) was inserted into Pichia pastoris wild type to generate a modified strain. The DNA was digested by Bam HI and transformed into PP1 (wild type Pichia pastoris strain). This was suitable as a host strain.

[00215] The kgtP gene was tagged with HA tag. The recombinant gene was detected by Western Blot using anti HA antibody. The band shown on the blot had the expected molecular weight for the kgtP gene.

EXAMPLE 6

Yeast modification to express the α-ketoglutarate transporter and produce hydroxylated collagen.

[00216] The host strain of example 5 is modified as described below:

The DNA encoding native Type III bovine collagen is sequenced (SEQ ID NO: 10) and the sequence is amplified by the PCR polymerase chain reaction protocol to create a linear DNA sequence.

[00217] DNA encoding P4HA / B enzymes is sequenced (SEQ ID NO: 8 and 9 (optimized for Pichia) or SEQ ID NO: 15 and 16 (native)) and the sequence is amplified by the PCR chain reaction protocol gives

Petition 870180132932, of 9/21/2018, p. 85/93 / 80 polymerase to create a linear DNA sequence.

[00218] The linear DNA containing the collagen sequence is inserted into a pichia genome and the linear DNA containing the P4HA / B sequences is inserted into the pichia genome.

[00219] DNA is transformed into the strain of example 5 using a Pichia Electroporation Protocol (Total System # 1652660 from Bio-Rad Gene Pulser XcellTM). Yeast cells are transformed with coexpression plasmid P4HA / B and transformants selected on Hygro plate (200 pg / ml).

[00220] A single colony of the strain resulting from example 1 is inoculated in 100 ml of YPD medium and grown at 30 degrees overnight with agitation at 215 rpm. The next day, when the culture reached OD600 ~ 3.5 (~ 3-5 X 10 ⁷ cells / OD600), it is diluted with fresh YPD to OD600 ~ 1.7 and grown for another hour at 30 degrees with agitation at 215 rpm.

[00221] The cells are rotated at 3,500g for 5 min; wash once with water and resuspend in 10 ml of 10 mM Tris-HCl (pH 7.5), 100 mM LiAc, 10 mM DTT (add fresh), 0.6 M sorbitol [00222] For each transformation, aliquot 8 X 10 ⁸ cells in 8 ml of 10 mM Tris-HCl (pH 7.5), 100 mM LiAc, 10 mM DTT, 0.6 M Sorbitol and incubate at room temperature for 30 min .

[00223] The cells are centrifuged at 5000g for 5 minutes and washed with 1.5 ml of ice-cold 1M Sorbitol 3 times and resuspended in 80 pl of ice-cold 1M Sorbitol [00224] Various amounts are added ( about 5 pg) of linearized DNA to the cells and mix by pipetting.

[00225] The cells and DNA mixture (80 to 100 pl) are added into a 0.2 cm cuvette and pulsed using the Pichia protocol (1500 v, 25 uF, 200Ω) [00226] immediately, cells within 1 ml of

Petition 870180132932, of 9/21/2018, p. 86/93 / 80 mixture of YPD and 1M Sorbitol (1: 1) and incubated at 30 degrees for> 2 hours [00227] The cells are plated in different densities.

[00228] Single colonies are inoculated in 2 mL of BMGY medium in a deep 24-well plate and cultured for at least 48 hours at 30 degrees Celsius with agitation at 900 rpm. The resulting cells were tested for collagen using cell lysis, SDS-page and pepsin assay following the procedure below.

[00229] Yeast cells were lysed in 1x lysis buffer using a Qiagen TissueLyser at a speed of 30 Hz continuously for 14 min. The lysis buffer was made up of 2.5 ml of 1 M HEPES (50 mM final concentration); 438.3 mg NaCl; final concentration 150 mM; 5 ml of glycerol; final concentration 10%; 0.5 ml of Triton X-100; final concentration 1%; and 42 ml of Millipure water.

[00230] The lysed cells are centrifuged at 2,500 rpm at 4 ° C for 15 minutes in a table centrifuge. The supernatant is retained and the pellet discarded.

[00231] The SDS-PAGE in the presence of 2-mercaptoethanol is performed on the supernatant, molecular weight markers, negative control and a positive control. After electrophoresis, the gel is removed and stained with Commassie Blue and then discolored in water.

[00232] A pepsin test is performed with the following procedure:

Before pepsin treatment, the BCA assay is performed to obtain the total protein for each sample according to the Thermo Scientific protocol. Total protein is normalized to the lowest concentration for all samples. (Note: if the lowest total protein concentration is less than 0.5mg / mL, do not use this concentration for normalization) [00233] 100 pL of lysate is placed in a microcentrifuge tube.

Petition 870180132932, of 9/21/2018, p. 87/93 / 80

A main mixture is created containing the following: 37% HCl (0.6pL of acid per 100 pL) and Pepsin (stock is 1mg / mL in deionized water and the final addition of pepsin should be in the proportion of 1:25 pepsin: total protein (weight: weight) After adding pepsin, mix 3X with a pipette and allow the samples to incubate for one hour at room temperature for the pepsin reaction to occur. ^ After one hour, add volume 1: 1 of LDS loading buffer containing β-mercaptoethanol for each sample and incubation for 7 minutes at 70 ° C is allowed. (In this situation, 100 pL of LDS must be added). Then, turn to 14,000 rpm for 1 minute to remove turbidity.

[00234] Add 18pL of the top of the sample in a 3-8% TAE buffer and run the gel for 1h 10 minutes at 150V. Amino acid analysis was performed to determine the percentage of hydroxylation. The expression of kgtP allows the transport of alpha ketoglutarate into the endoplasmic reticulum, which results in an increase in collagen and the level of hydroxylated collagen.

EXAMPLE 7 The yeast of Example 1 with the ascorbate pathway is modified with the procedure of Example 5 to transform the carrier into the yeast that supplies Pichia with both the ascorbatic pathway and the kgtP transporter.

EXAMPLE 8 [00236] The yeast of Example 7 with the ascorbate route and the carrier for kgtP are modified with the procedure of Example 2 to provide Pichia with the ascorbate route, the carrier for kgtP and hydroxylated collagen.

EXAMPLE 9 [00237] The gene for A L-gulono-1,4-lactone oxidase (acquired from Eurofins Genomics) was codon-optimized for expression in Pichia (SEQ

Petition 870180132932, of 9/21/2018, p. 88/93 / 80

ID NO 17). The gene was cloned under the constitutive promoter pDF and the antibiotic marker used was zeocin. The plasmid was transformed into Pichia cells using the same electroporation method as Example 2 and placed on YPD plates with 50pg / ml of zeocin. The plates were incubated at 30 ⁰ C for 2 days or until transformants start to appear on the plate. [00238] Six transformants were chosen and grown plate into 2 ml BMGY medium in 24-well plates for 18 hours with constant stirring at 30 ⁰ C. After 18 hours, varied concentration of the substrate (Lgulono-1,4-lactone ) was added to the growing culture (0, 0.1 and 1 g / L final concentration). Wild-type Pichia cells were used as a control for the experiment and the substrate was also added to the control culture. Cultures were harvested 20 hours after adding the substrate. The optical density was normalized and the cells were lysed in water by mechanical shear. Two methods were used to quantify the concentration of ascorbic acid in the cell lysate. In the first method, an abcam kit (Cat # ab65356) was used, in which the fluorescence intensity of the reaction mixture was measured. The second method is known as the Sullivan and Clarke method, in which the absorbance for samples other than cell lysates was recorded as the measure of ascorbic acid production. The cell lysates of the transformants with the L-gulono-1,4lactone-oxidase gene showed greater fluorescence intensity and greater absorbance than the controls (wild type Pichia and Transformants grown without the substrate). Preliminary data indicated that ascorbic acid was made when 1 g / L of the substrate was added to the cells and ~ 10 nmol of ascorbic acid was produced at later times (36 hours after adding the substrate).

Claims (14)

1. Recombinant yeast cell, characterized by the fact that it comprises a polynucleotide that encodes an α-ketoglutarate transporter. 2. Recombinant yeast cell according to claim 1, characterized by the fact that the α-ketoglutarate transporter has a sequence that is at least 90% identical to SEQ ID NO: 1. 3. Recombinant yeast cell according to claim 1, characterized by the fact that the polynucleotide is optimized for expression in said yeast cell. 4. Recombinant yeast cell according to claim 1, characterized by the fact that the polynucleotide is recombined in the yeast cell genome or is present in an episomal way in a recombinant vector that includes transcriptional control elements that allow the production of a coding sequence mRNA in the yeast cell. Recombinant yeast cell according to claim 1, characterized in that it additionally comprises at least one polynucleotide encoding collagen or a fragment of collagen thereof. 6. Recombinant yeast cell according to claim 1, characterized in that it additionally comprises at least one polynucleotide encoding prolyl-4-hydroxylase, prolyl-3hydroxylase and lysyl hydroxylase. 7. Recombinant yeast cell according to claim 1, characterized by the fact that the collagen or a fragment of collagen thereof is glycosylated to at least one hydroxyl residue. 8. Recombinant yeast cell according to Petition 870180132932, of 9/21/2018, p. 90/93 2/2 claim 1, characterized by the fact that it additionally comprises at least one polynucleotide encoding a glycosyl hydroxylase. 9. Recombinant yeast cell according to claim 1, characterized by the fact that the yeast cell is Pichia pastoris. 10. Method for producing recombinant yeast cell as defined in claim 1, the method characterized by the fact that it comprises transforming the yeast cell with the polynucleotide encoding an α-ketoglutarate transporter that is operationally linked to one or more control elements transcriptional that allows the production of a coding sequence mRNA in the yeast cell. 11. Method for producing hydroxylated collagen in a yeast cell, the method characterized by the fact that it comprises culturing the recombinant yeast cell as defined in claim 1, in a medium. Method according to claim 11, characterized in that it further comprises isolating or purifying the modified recombinant collagen produced by the recombinant yeast cell in the culture. 13. Collagen, characterized by the fact that it is obtained by the method as defined in claim 11. 14. Biofabricated leather material, characterized by the fact that it comprises collagen as defined in claim 13.